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Phosphoproteomic Profiling Reveals Vasopressin- Regulated Phosphorylation Sites in Collecting Duct

Amar D. Bansal,* Jason D. Hoffert,* Trairak Pisitkun,* Shelly Hwang,* Chung-Lin Chou,* Emily S. Boja,† Guanghui Wang,† and Mark A. Knepper*

*Epithelial Systems Biology Laboratory, and †Proteomics Core Facility, Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland

ABSTRACT Protein phosphorylation is an important component of vasopressin signaling in the renal collecting duct, but the database of known phosphoproteins is incomplete. We used tandem mass spectrometry to identify vasopressin-regulated phosphorylation events in isolated rat inner medullary collecting duct (IMCD) suspensions. Using multiple search algorithms to identify the phosphopeptides from spectral data, we expanded the size of the existing collecting duct phosphoproteome database from 367 to 1187 entries. Label-free quantification in vasopressin- and vehicle-treated samples detected a significant change in the phosphorylation of 29 of 530 quantified phosphopeptides. The targets include important structural, regulatory, and transporter proteins. The vasopressin-regulated sites included two known sites (Ser-486 and Ser-499) present in the urea channel UT-A1 and one previously unknown site (Ser-84) on vasopressin-sensitive urea channels UT-A1 and UT-A3. In vitro assays using synthetic peptides showed that purified protein kinase A (PKA) could phosphorylate all three sites, and immunoblotting confirmed the PKA dependence of Ser-84 and Ser-486 phosphorylation. These results expand the known list of collecting duct phosphoproteins and highlight the utility of targeted phosphoproteomic approaches.

J Am Soc Nephrol 21: 303–315, 2010. doi: 10.1681/ASN.2009070728

Vasopressin plays a central role in collecting duct In a previous study,17 we used tandem mass physiology. Signaling through the V2 receptor re- spectrometry (LC-MS/MS)-based quantitative sults in an increase of cAMP levels and causes acti- phosphoproteomics to partially annotate the phos- vation of protein kinase A (PKA).1,2 In addition, phoproteome of rat inner medullary collecting duct over 200 other serine/threonine protein kinases are (IMCD). We subsequently quantified the differen- expressed in native collecting duct cells,3 and some tial phosphorylation of four serine residues (Ser- of these have been shown to play important roles in 256, Ser-261, Ser-264, and Ser-269) in the C-termi- the response to vasopressin.4–10 Vasopressin signal- nal tail of rat aquaporin-2 (AQP2) in response to ing is important not only for regulation of water short-term exposure to the vasopressin analog transport through aquaporins11 but also for regula- dDAVP.17,18 We also found a number of phosphor- tion of urea12 and sodium transport.13,14 Vasopres- sin also regulates long-term gene expression of col- Received July 15, 2009. Accepted November 2, 2009. lecting duct proteins, such as aquaporins.15,16 Because protein phosphorylation plays a central Published online ahead of print. Publication date available at www.jasn.org. role in vasopressin signaling, the identification and quantification of phosphorylated proteins in re- Correspondence: Dr. Mark A. Knepper, National Institutes of Health, 10 Center Drive, Building 10, Room 6N260, Bethesda, sponse to vasopressin are essential to understand- MD 20892-1603. Phone: 301-496-3064; Fax: 301-402-1443; E-mail: ing the mechanism of action of this hormone in [email protected] collecting duct. Copyright ᮊ 2010 by the American Society of Nephrology

J Am Soc Nephrol 21: 303–315, 2010 ISSN : 1046-6673/2102-303 303 BASIC RESEARCH www.jasn.org

A BC

IMCD isolate IMCD isolate IMCD isolate

dDAVP treatment dDAVP treatment control dDAVP treatment control

Protein isolation Protein isolation Protein isolation Protein isolation Protein isolation Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis

SCX fractionation

IMAC IMAC IMAC IMAC IMAC

LC-MS/MS Profiling large-scale quantification large-scale quantification TIS quantification TIS quantification

Figure 1. The three different LC-MS/MS experiments performed are as follows: (A) The initial phosphoproteomic profiling experiment consisted of a single dDAVP-treated sample that was processed using SCX fractionation. (B) The second experiment was performed for large-scale quantification using a nonselective “profiling” mode. (C) The third experiment was performed for targeted quantification by selection of precursor ion m/z ratios, so-called targeted ion selection (TIS) mode. ylation sites on the vasopressin-sensitive urea channel, UT- dDAVP-treated IMCD sample (Figure 1A) as described in A.17 However, because of the limited sensitivity of the experi- “Concise Methods,” followed by phosphopeptide enrichment mental approach, we were unable to quantify changes in via immobilized metal affinity chromatography (IMAC). Sam- phosphorylation at these sites in UT-A, despite evidence for ples representing 24 SCX fractions were analyzed on a Thermo such sites on the basis of previous studies.19,20 LTQ mass spectrometer, and the resulting spectra were One of the primary aims of this study was to increase the searched using three different search algorithms: SEQUEST, sensitivity of our MS-based workflow to annotate a larger por- InsPecT, and OMSSA. Figure 2 shows a Venn diagram of tion of the IMCD phosphoproteome. This was accomplished unique phosphopeptides identified from each of the three in three ways: (1) by implementing an effective, chromatogra- search algorithms. All datasets were filtered for a Ͻ2% false- phy-based stratification technique for our peptide samples, (2) discovery rate on the basis of target-decoy analysis.21 Overlap- by using a higher resolution mass spectrometer, and (3)by using multiple proteomic search algorithms to process the MS OMSSA InsPecT data. Using these combined approaches, we increased coverage 1322 2120 of the IMCD phosphoproteome by approximately 3-fold com- pared with the previous study.17 In addition, we present large- scale phosphoproteomic data quantifying the effect of vaso- 206 pressin on phosphorylation of IMCD proteins. Last, we 279 identify and quantify six phosphorylation sites on the vaso- pressin-sensitive urea channel (isoforms A1 and A3) and dem- 948 735 onstrate that three of these sites undergo large increases in 102 phosphorylation in response to short-term dDAVP treatment. 231 206 RESULTS SEQUEST Phosphoproteomic Profiling of IMCD 1274 Our previous study using an LC-MS/MS “shotgun” approach identified 223 unique phosphoproteins in native IMCD cells.17 One of the aims of this study was to expand the size of the Combined total 2707 identified collecting duct phosphoproteome by adding meth- Figure 2. Venn diagram shows the number of unique phos- ods that would increase overall sensitivity; viz., using sample phopeptide identifications that resulted from each of three search fractionation via strong cation exchange (SCX) and employing algorithms (SEQUEST, InsPecT, and OMSSA). A false-discovery multiple MS search algorithms. SCX was performed on a single rate stringency of Ͻ2% was used for each of the searches.

304 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 303–315, 2010 www.jasn.org BASIC RESEARCH

kDa control dDAVP (10-9 M) mentation (Figure 1B). All 10 samples were searched with SE- QUEST (false-discovery rate Ͻ2%), and relative quantifica- 37 AQP2 22 Ser(p)-256 tion was performed using QUOIL. A total of 530 unique 25 phosphopeptides from 278 proteins were quantified (Figure

4). For statistical analysis, values were converted to log2 of the Figure 3. Immunoblot showing that dDAVP increased phosphor- ratio (D/C), where D and C refer to the normalized areas of the ylation of Ser-256 of AQP2. An immunoblot of IMCD protein extracted peptide ion chromatograms under dDAVP-treated isolate shows five pairs of control and dDAVP-treated samples. and control conditions, respectively. Brackets in Figure 4 indi- Bands at 29 and 37 kDa indicate Ser(p)-256 bands (nonglycosy- cate the subset of 54 phosphopeptides with log (D/C) values lated and glycosylated forms, respectively). 2 either greater than 0.58 or less than Ϫ0.58. Of these 54 phos- ping regions consist of phosphopeptides that were identified phopeptides, 29 represented phosphopeptides with statisti- by more than one search algorithm and indicate a match not cally significant (P Ͻ 0.05) changes in abundance between the only of the phosphopeptide primary amino acid sequence but five paired control and dDAVP-treated IMCD tubules. These also for the exact site(s) of phosphorylation. A total of 735 phosphopeptides are shown in Table 1 and are marked with a phosphopeptides were identified by all three search algorithms superscript letter a. Phosphopeptides whose abundance did (as shown by the central overlap region). InsPecT identified not change significantly are marked with a superscript letter c the largest number of unique phosphopeptides (2120), with and were included as an internal negative control showing that the highest number of identifications that were not shared with the changes in phosphopeptide abundances found are most either of the other two programs (948). Annotated phos- likely not due to changes in the amount of protein analyzed. phopeptide data from all three searches are accessible online at Supplementary Table 1 is a complete list of phosphopeptides Ϫ the Collecting Duct Phosphoprotein Database (http://dir. with mean log2(D/C) values between 0.58 and 0.58. nhlbi.nih.gov/papers/lkem/mpkccdprot/). This study in- creases the size of the Collecting Duct Phosphoprotein Data- Regulation of ␤-Catenin Phosphorylation by dDAVP base from 367 unique phosphoproteins to 1187 unique phos- One of the phosphorylation sites that increased in response to phoproteins. dDAVP was Ser-552 of ␤-catenin (Table 1, part B). Because ␤-catenin has been proposed to play a role in regulation of Verification of IMCD Response to dDAVP aquaporin-2,23 we investigated this response further using a After profiling the dDAVP-treated IMCD phosphopro- phosphospecific antibody. Figure 5A shows an immunoblot teome, we performed large-scale profiling experiments on performed on protein isolates from the same five pairs of ani- paired vehicle and dDAVP-treated IMCD samples to deter- mals used in our large-scale quantitation experiment. The blot mine which phosphoproteins were regulated by vasopres- was probed with an antibody recognizing Ser(p)-552-␤-cate- sin. Phosphorylation on Ser-256 of AQP2 occurred in re- nin. As shown in Figure 5A, Ser(p)-552-␤-catenin was in- sponse to short-term vasopressin exposure,17 and creased 3-fold in response to dDAVP (band densities: vehicle, immunoblotting with a phosphospecific antibody was used 4.8 Ϯ 0.2; dDAVP, 14.8 Ϯ 0.3; P Ͻ 0.05). An immunoblot for to verify the expected increase in Ser(p)-256 abundance in total ␤-catenin was also performed and shows that dDAVP- vasopressin-treated samples. Figure 3 shows that dDAVP increased the amount of Ser(p)-256-AQP2 compared with con- Distribution of phosphopeptides quantified by LC-MS/MS trol samples (bands at 29 and 37 kDa). 350 Total protein abundance of AQP2 does not change with dDAVP treatment, as 300 283 18 shown by Hoffert et al. 250 193 Large-Scale Quantitative 200

Phosphoproteomics Reveals Potential Count 150 Downstream Targets of Vasopressin Table 1A (28 phosphopeptides) Table 1B (26 phosphopeptides) 100 Signaling 50 21 We isolated IMCD suspensions and pre- 10 0 1 2 0004 4 320 322 pared five paired control and dDAVP- 0 treated samples all from different rats. -1.0-1.5-2.0-2.5-3.0-3.5-4.0-4.5 0-0.5 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 Samples were digested with trypsin; phos- Range of mean log 2(D/C) phopeptide enrichment was performed us- Figure 4. Histogram showing distribution of the mean log2(D/C) for all peptides ing IMAC; and the samples were analyzed quantified using LC-MS/MS. The vast majority of peptides did not change with dDAVP Ϫ on the LTQ-Orbitrap MS platform using treatment (476 (90.0%) of 530), having mean log2(D/C) values between 0.58 and collision-induced dissociation (CID) frag- 0.58.

J Am Soc Nephrol 21: 303–315, 2010 Phosphoproteomics of IMCD 305 BASIC RESEARCH www.jasn.org

Table 1. List of phosphopeptides that show significant change in abundance or no significant change with dDAVP treatment compared with control samples Mean Log No. Protein Name Accession No. Sequence (Charge) 2 Sites D/C (D/C) Part A 1 aquaporin 2 (collecting duct) NP࿝037041 R.QSVELHS*PQSLPR.G(ϩ2) 0.06 Ϫ3.95 Ϯ 0.24a S261 2 aquaporin 2 (collecting duct) NP࿝037041 R.RQSVELHS*PQSLPR.G(ϩ3) 0.10 Ϫ3.39 Ϯ 0.13a S261 3 aquaporin 2 (collecting duct) NP࿝037041 R.QSVELHS*PQSLPR.G(ϩ3) 0.11 Ϫ3.20 Ϯ 0.15a S261 4 ArfGAP with FG repeats 1 NP࿝001129068 K.SLLGESAPALHLNKGT*PT* 0.34 Ϫ1.55 Ϯ 0.27a T177,b T179b QSPVVGR.S(ϩ3) 5 septin 9 isoform 2 NP࿝789826 R.LVDTLSQRS*PKPSLR.R(ϩ3) 0.41 Ϫ1.30 Ϯ 0.08a S67b 6 ArfGAP with FG repeats 1 NP࿝001129068 K.SLLGESAPALHLNKGT*PT- 0.44 Ϫ1.19 Ϯ 0.21a T177, S181 QS*PVVGR.S(ϩ3) 7 ysv oncogene homolog NP࿝110484 R.TIYVRDPT*S*NKQQRPVP- 0.50 Ϫ1.01 Ϯ 0.37c T37,b S38,b isoform A ES*QLLPGQR.F(ϩ3) S48b 8 (ABC1), member 1 NP࿝835196 K.HVKAEMEQMALDVGLP- 0.50 Ϫ1.00 Ϯ 0.49c S964 PS*KLK.S(ϩ3) 9 tensin like SH2 domain XP࿝341257 R.WDSY*ENMSADGEVLHT- 0.51 Ϫ0.98 Ϯ 1.12c Y410b containing 1 QGPVDGSLYAK.V(ϩ3) 10 hsp 90, alpha (cytosolic), NP࿝786937 R.DKEVS*DDEAEEKEEK. 0.52 Ϫ0.93 Ϯ 0.82c S231 class A member 1 E(ϩ3) 11 calpastatin isoform a NP࿝445747 K.NEAITGPLPDS*PKPMGID- 0.55 Ϫ0.86 Ϯ 1.15c S260,b S280b HAIDALSSDFTCS*SPTGK. Q(ϩ3) 12 calpastatin isoform a NP࿝445747 K.NEAITGPLPDS*PKPMGID- 0.55 Ϫ0.86 Ϯ 1.15c S260,b S281b HAIDALSSDFTCSS*PTGK. Q(ϩ3) 13 calpastatin isoform a NP࿝445747 K.NEAITGPLPDS*PKPMGID- 0.56 Ϫ0.84 Ϯ 1.13c S260,b T283b HAIDALSSDFTCSSPT*GK. Q(ϩ3) 14 tensin XP࿝237286 R.HPVGSHQVPGLHSGVVTT- 0.60 Ϫ0.73 Ϯ 0.43c S1523b PGS*PSLGR.H(ϩ4) 15 tx elongation factor A (SII) 1 XP࿝001059640 K.KKEPAISSQNS*PEAR. 0.62 Ϫ0.70 Ϯ 0.77c S100b isoform 2 E(ϩ3) 16 tight junction protein 2 NP࿝446225 R.SQEES*PVPQPR.T(ϩ2) 0.62 Ϫ0.69 Ϯ 0.09a S463 17 nuclear RNA export factor 3 XP࿝001053711 K.S*SNINS*ILELFPK.L(ϩ2) 0.62 Ϫ0.69 Ϯ 0.23a S287,b S292b 18 PDLIM1 interacting kinase 1 NP࿝001101454 R.LDTS*DLEPT*LKVADFGLS- 0.62 Ϫ0.68 Ϯ 0.86c S180,b T185,b like KVCS*ASGQNPEEPVSVNK. S198b C(ϩ3) 19 (urea transporter), member 2 NP࿝808877 K.LYESELSSPT*WPSSSQDTH- 0.63 Ϫ0.67 Ϯ 0.93c T37b isoform 2 PALPLLEMPEEK.D(ϩ3) 20 epsin 3 NP࿝001019962 R.TPVLPSGPPITDPWAPSSPT* 0.63 Ϫ0.66 Ϯ 0.57c T361b PK.L(ϩ2) 21 (urea transporter), member 2 NP࿝808877 K.DLRS*S*DEDSHIVK.I(ϩ3) 0.63 Ϫ0.66 Ϯ 0.59c S62, S63 isoform 2 22 tensin XP࿝237286 K.VSS*SPVANGMAS*PSGSST- 0.63 Ϫ0.66 Ϯ 0.43c S1667,b VSFSHTLPDFSK.Y(ϩ3) S1676b 23 calnexin NP࿝742005 K.SDAEEDGGTGS*QDEEDSK- 0.64 Ϫ0.65 Ϯ 0.59c S563b PK.A(ϩ3) 24 AHNAK nucleoprotein XP࿝001078032 K.LEGEIKVPDVDISS*PGVNVE- 0.64 Ϫ0.65 Ϯ 0.19c S1807b isoform 1 APDIHVK.A(ϩ4) 25 prostaglandin E synthase 3 NP࿝001124461 K.DWEDDS*DEDMSNFDR. 0.64 Ϫ0.64 Ϯ 1.10c S113b (cytosolic) F(ϩ2) 26 beta-2-syntrophin XP࿝001071043 R.GLGPPS*PPAPPR.G(ϩ2) 0.64 Ϫ0.64 Ϯ 0.37c S120 27 PC4 and SFRS1 interacting NP࿝786941 K.NLAKPGVT*STSDS*EEDDD- 0.66 Ϫ0.60 Ϯ 1.07c T269,b S274b protein 1 QEGEK.K(ϩ3) 28 tight junction protein 2 NP࿝446225 R.KVQVAPLQGS*PPLSHDDR. 0.66 Ϫ0.59 Ϯ 0.24c S107b G(ϩ3)

306 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 303–315, 2010 www.jasn.org BASIC RESEARCH

Table 1. (Continued) Mean Log No. Protein Name Accession No. Sequence (Charge) 2 Sites D/C (D/C) Part B 1 AHNAK nucleoprotein XP࿝001078032 R.NRS*NS*FSDEREFSAPST- 1.51 0.59 Ϯ 0.25c S2454,b isoform 1 PTGTLEFAGGEGK.G(ϩ3) S2456b 2 AHNAK nucleoprotein XP࿝001078032 R.NRSNS*FS*DEREFSAPSTP- 1.51 0.59 Ϯ 0.25c S2456,b isoform 1 TGTLEFAGGEGK.G(ϩ3) S2458b 3 leucine-rich repeat (in FLII) NP࿝001019932 R.RGSGDTSS*LIDPDTSLSE- 1.55 0.63 Ϯ 0.29c S138b interacting protein 2 LR.E(ϩ3) 4 aquaporin 2 (collecting duct) NP࿝037041 R.RRQS*VELHS*PQSLPR. 1.55 0.63 Ϯ 0.06a S256, S261 G(ϩ3) 5 tankyrase 1-binding protein XP࿝215763 K.RAS*VSTNQNTDENDQELR. 1.67 0.74 Ϯ 0.18a S883b of 182 kDa M(ϩ3) 6 aquaporin 2 (collecting duct) NP࿝037041 R.RQS*VELHS*PQSLPR.G(ϩ2) 1.73 0.79 Ϯ 0.17a S256, S261 7 PCTAIRE protein kinase 3 NP࿝001093976 R.RFS*MEDLNKR.L(ϩ3) 1.77 0.82 Ϯ 0.05a S66b 8 AHNAK nucleoprotein XP࿝001078032 R.NRS*NS*FSDEREFSAPSTPT- 1.91 0.93 Ϯ 0.41c S2454,b isoform 1 GTLEFAGGEGK.G(ϩ4) S2456b 9 aquaporin 2 (collecting duct) NP࿝037041 R.RRQS*VELHS*PQSLPR. 1.93 0.95 Ϯ 0.21a S256, S261 G(ϩ2) 10 calcium regulated heat NP࿝690003 R.DRS*PS*PLRGNVVPSPLPTR. 2.28 1.19 Ϯ 0.27a S30, S32 stable protein 1 R(ϩ3) 11 desmoplakin isoform I XP࿝225259 R.SMS*FQGIR.Q(ϩ2) 2.28 1.19 Ϯ 0.77c S2216b isoform 2 12 PDZ and LIM domain 5 NP࿝445778 R.RGS*QGDIKQQNGPPR. 2.64 1.40 Ϯ 0.42a S228 K(ϩ3) 13 beta-catenin NP࿝445809 R.RTS*MGGTQQQFVEGVR. 2.69 1.43 Ϯ 0.04a S552b M(ϩ3) 14 beta-catenin NP࿝445809 R.RTSMGGT*QQQFVEGVR. 2.69 1.43 Ϯ 0.04a T556b M(ϩ3) 15 PCTAIRE protein kinase 3 NP࿝001093976 R.RFS*MEDLNK.R(ϩ2) 3.05 1.61 Ϯ 0.21a S66 16 beta-catenin NP࿝445809 R.RTS*MGGTQQQFVEGVR. 3.29 1.72 Ϯ 0.19a S552b M(ϩ2) 17 aquaporin 2 (collecting duct) NP࿝037041 R.RQS*VELHS*PQS*LPR. 3.92 1.97 Ϯ 0.07a S256, S261, G(ϩ3) S264 18 aquaporin 2 (collecting duct) NP࿝037041 R.RRQS*VELHSPQS*LPR. 4.14 2.05 Ϯ 0.13a S256,b S264b G(ϩ3) 19 plexin domain containing 2 NP࿝001101892 R.RGS*GHPAYAEVEPVGEK- 4.38 2.13 Ϯ 0.24a S507 EGFIVSEQC.-(ϩ3) 20 aquaporin 2 (collecting duct) NP࿝037041 R.RRQS*VELHSPQSLPR. 9.00 3.17 Ϯ 0.20a S256 G(ϩ3) 21 aquaporin 2 (collecting duct) NP࿝037041 R.RRQS*VELHS*PQS*LPR. 9.38 3.23 Ϯ 0.39a S256, S261, G(ϩ2) S264 22 kinesin 13B NP࿝998791 R.RRS*S*GLQPQGAPEAR. 10.93 3.45 Ϯ 0.20a S1718, S1719 R(ϩ3) 23 aquaporin 2 (collecting duct) NP࿝037041 R.RQS*VELHS*PQS*LPR. 12.30 3.62 Ϯ 0.41a S256, S261, G(ϩ2) S264 24 aquaporin 2 (collecting duct) NP࿝037041 R.RQS*VELHSPQSLPR. 14.93 3.90 Ϯ 0.52a S256 G(ϩ2) 25 (urea transporter), member 2 NP࿝062220 R.RKS*VFHIEWSSIR.R(ϩ4) 17.51 4.13 Ϯ 0.22a S486 isoform 1 26 (urea transporter), member 2 NP࿝062220 R.RKS*VFHIEWSSIR.R(ϩ3) 19.84 4.31 Ϯ 0.31a S486 isoform 1

Mean and SE values are calculated from log2(D/C), where D and C are peak areas for dDAVP-treated and control samples, respectively. Phosphorylation sites are indicated with an asterisk. aP Ͻ 0.05; significant change in abundance compared with control samples. bAmbiguity in phosphorylation site assignment by the Ascore algorithm, despite certainty in phosphopeptide identification. cNo significant change in abundance compared with control samples.

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kDa control dDAVP (10-9 M)

117 UT-A1 97

47 37 AQP-2 Ser(p)-256 25

Figure 6. Immunoblot shows that dDAVP treatment of IMCD does not result in increased total protein abundance of UT-A1. Immunoblot shows three pairs of control and dDAVP-treated samples. Proteins at 29 and 37 kDa are Ser(p)-256-AQP2 (nong- Figure 5. dDAVP increases ␤-catenin phosphorylation in rat lycosylated and glycosylated forms, respectively). Protein at 97 IMCD. (A) Top panel: Immunoblot of IMCD suspensions showing and 117 kDa is UT-A1. five pairs of control and dDAVP-treated samples. Band at 97 kDa is Ser(p)-552-␤-catenin. dDAVP causes increased phosphorylation of Ser-552 of ␤-catenin. Bottom panel: Immunoblot of IMCD and treated samples (P ϭ 0.11). The same immunoblot was suspensions showing the same five pairs of control and dDAVP- also probed with a phosphospecific antibody recognizing treated samples using antibody recognizing total ␤-catenin. (B) Ser(p)-256 on AQP2 to verify the responsiveness of IMCD Top panel: Immunoblot of whole inner medulla from Brattleboro tubules to dDAVP stimulation. ␤ rats showing increased Ser(p)-552- -catenin in response to From our SCX profiling data, we identified phosphoryla- dDAVP given intramuscularly. Bottom panel: Immunoblot of tion of the vasopressin-sensitive urea channel (isoforms A1 whole inner medulla from Brattleboro rats showing that there is and A3) on Ser-10, Ser-62, Ser-63, Ser-84, and Ser-486 (http:// no increase in total ␤-catenin abundance in response to dDAVP dir.nhlbi.nih.gov/papers/lkem/mpkccdprot/). Of these five given intramuscularly. sites, Ser-10 and Ser-84 have not been previously reported. Ser-486 was identified by Hoffert et al.17 using a similar ap- treatment does not (P Ͼ 0.40) increase total protein abun- proach and was subsequently found to be functionally impor- dance (Figure 5A, bottom). Thus, we were able to show con- tant for urea transport activity by Blount et al.20 The same vergent results for increased ␤-catenin phosphorylation on study also demonstrated the importance of phosphorylation at Ser-552 in response to dDAVP treatment using both LC- Ser-499 for the transport function of UT-A1.20 For this study, a MS/MS and immunoblotting. trypsin digest could not be used to identify the peptide con- To address whether a similar change in ␤-catenin phos- taining Ser-499, because the resulting peptide was too short for phorylation occurs in the inner medulla of intact kidney, LC-MS/MS detection. Alternatively, sample digestion with Brattleboro rats were injected intramuscularly with dDAVP or chymotrypsin was predicted to produce a peptide of suitable vehicle, and in 45 minutes, the whole inner medulla was iso- length. However, attempts to identify the Ser-499-containing lated for immunoblot analysis. As shown in Figure 5B, Ser(p)- chymotryptic peptide were precluded by poor fragmentation 552-␤-catenin was increased nearly 2-fold in response to of the parent ion using CID as the fragmentation technique. dDAVP (band densities: vehicle, 10.9 Ϯ 1.1; dDAVP, 20.1 Ϯ Therefore, we used an alternative fragmentation technique, 2.6; P Ͻ 0.05). electron transfer dissociation (ETD), to successfully identify the chymotryptic peptide containing Ser-499 (Figure 7C), and dDAVP Increases Phosphorylation of the Vasopressin- CID was used for the remaining sites (Ser-10, Ser-62/63, Ser- Sensitive Urea Channel 84, and Ser-486). Before examining the effect of short-term dDAVP treatment On the basis of phosphorylation sites identified from our on UT-A phosphorylation, it was necessary to establish that profiling data and evidence showing that Ser-499 is function- total UT-A protein abundance does not change as a result of ally important for urea transport,20 we used targeted selection short-term dDAVP treatment, and thus was not contributing of precursor ion masses, so-called targeted ion selection (TIS) to changes in abundance of the UT-A phosphopeptides. The (Figure 1C), to quantify changes in phosphorylation in re- immunoblot in Figure 6 was probed with an antibody recog- sponse to dDAVP for each of the sites. dDAVP treatment re- nizing total UT-A1.24 The 97- and 117-kD bands represent sulted in no change in phosphorylation at Ser-10 (Supplemen- UT-A1. There was no significant difference between control tary Figure 1) or phosphorylation at both Ser-62 and Ser-63

308 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 303–315, 2010 www.jasn.org BASIC RESEARCH

A Ser(p)-84, UT-A1/A3 reconstructed ion chromatograms were used to calculate changes in abundances of 72.8 phosphorylated peptides in response to dDAVP. Of these sites, only Ser-84 is control shared by UT-A1 and UT-A3.

18.2 Protein Kinase A Can Directly

72.8 Phosphorylate Ser-84, Ser-486, and

Signal Intensity (x10,000) Ser-499 of UT-A in Vitro

dDAVP UT-A1 contains several putative PKA 25–27 18.2 phosphorylation motifs, including Ser-84, Ser-486, and Ser-499. To determine Peptide Sequence log2(D/C) D/C p n 21.80 23.10 24.14 Time (min) R.RES*ELPR.R 2.55 + 0.07 5.86 <0.001 6 whether PKA can, in fact, phosphorylate these three sites, we performed in vitro as- B Ser(p)-486, UT-A1 says using purified PKA catalytic subunit and synthetic UT-A peptides as substrates. 23.2 Figure 8A shows phosphorylation on the C-terminal tail of aquaporin-2 on Ser-256, control which served as a positive control. In addi- 5.8 tion, PKA also was able to phosphorylate peptides containing Ser-486, Ser-84, and 23.2 Ser-499 sites on UT-A1 (Figure 8, B, C, and Signal Intensity (x10,000) D, respectively). dDAVP 5.8 Phosphorylation of Ser-84 in UT-A1/3 Peptide Sequence log2(D/C) D/C p n and Ser-486 in UT-A1 Is Increased by 30.7031.95 32.95 R.RKS*VFHIEWSSIR.R 2.32 + 0.28 4.99 <0.005 5 Time (min) dDAVP and Is Blocked by Inhibition of PKA C Ser(p)-499, UT-A1 To test whether PKA regulates phosphory-

806 lation of UT-A1 and UT-A3 in vivo, iso- lated rat IMCD samples were incubated control control with dDAVP in the presence or absence of ␮ 201 10 M PKA inhibitor H89, followed by im- munoblotting with newly generated anti-

806 bodies that recognize phosphorylated

Signal Intensity (x10,000) UT-A1 and UT-A3 at Ser-84, and phos- dDAVP phorylated UT-A1 at Ser-486 (Figure 9A). dDAVP Phosphorylation at both Ser-84 and 201 Peptide Sequence log2(D/C) D/C p n W.SSIRRRS*KVF.G 2.19 + 0.30 4.56 <0.02 3 Ser-486 was significantly reduced in the 12.20 13.45 14.70 Time (min) presence of H89, suggesting that PKA may directly or indirectly regulate phosphoryla- Figure 7. MS quantification of UT-A phosphorylation is illustrated by reconstructed tion at these sites in response to short-term peptide ion chromatograms from QUOIL software. The data show an increase in peak areas with dDAVP treatment compared with control for (A) Ser(p)-84 on both UT-A1 dDAVP treatment (Figure 9, A, C, D, and and UT-A3, (B) Ser(p)-486 on UT-A1, and (C) Ser(p)-499 on UT-A1. Also shown in each E). Inhibition of Ser(p)-256-AQP2 was subfigure are mean and SE values for all quantifications along with a representative used as a positive control to demonstrate MS2 spectrum for the identified peptide. that the H89 drug was working (Figure 9, A and B). (Supplementary Figure 2). However, phosphorylation at Ser- A recent study has also implicated the MEK-ERK pathway 84, Ser-486, and Ser-499 increased significantly (P Ͻ 0.02) in in regulating UT-A phosphorylation in response to forskolin response to dDAVP. Figure 7 shows representative recon- in rat IMCD.28 To test whether extracellular signal-regulated structed ion chromatograms for Ser-84, Ser-486, and Ser-499 kinase (ERK) mitogen-activated protein (MAP) kinase is in- phosphopeptides analyzed by LC-MS/MS. Each of the three volved in regulating UT-A phosphorylation, isolated rat IMCD subfigures also contains a representative MS2 fragmentation samples were incubated with dDAVP in the presence or ab- spectrum and a table summarizing all measurements for the sence of 10 ␮M MEK1/2 (map kinase kinase 1 and 2) inhibitor listed peptide. The ratios of the integrated peak areas from U0126. Immunoblotting for phosphorylated ERK (pERK)

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A B C D Ser(p)-256, AQP2 Ser(p)-486, UT-A1 Ser(p)-84, UT-A1/A3 Ser(p)-499, UT-A1

1160 17.6 39.2 198

290 - PKA 4.4 - PKA 9.8 - PKA 49.6 - PKA

1160 17.6 39.2 198

Signal Intensity (x10,000) 290 + PKA 4.4 + PKA 9.8 + PKA 49.6 + PKA

Time (min) Time (min) Time (min) Time (min)

Figure 8. PKA can phosphorylate Ser-84 (UT-A1 and UT-A3), Ser-486-UT-A1, and Ser-499-UT-A1 in vitro. In each of the windows, the top half shows the reconstructed ion chromatogram for the phosphopeptide when PKA was not present in the reaction mixture; the bottom half shows the corresponding profiles for the phosphopeptide when PKA was added to the reaction mixture. (A) The C-terminal tail of AQP-2, which served as a positive control, is phosphorylated by PKA on Ser-256. Phosphorylation occurred only in the presence of kinase for (B) Ser-486-UT-A1, (C) Ser-84 (UT-A1 and UT-A3), and (D) Ser-499-UT-A1. demonstrated that dDAVP treatment did not increase ERK MS/MS (Figure 6) and immunoblot (Figure 9) analysis. In a 20 phosphorylation and that U0126 significantly blocked ERK previous study using transfected LLC-PK1 cells, Blount et al. phosphorylation in both control and dDAVP-treated samples showed that S486A and S499A mutants each had reduced 32P (Figure 9, A and F). U0126 also produced a slight but signifi- incorporation into UT-A1 as well as attenuated urea flux in cant reduction in UT-A1 phosphorylated at Ser-84 in dDAVP- response to forskolin treatment. The importance of these two treated samples (Figure 9C). However, inhibition of the ERK sites was highlighted by the fact that the S486A/S499A double pathway failed to significantly affect the level of AQP2 phos- mutants did not show any significant urea flux increase in re- phorylated at Ser-256, UT-A3 phosphorylated at Ser-84, and sponse to forskolin. Because of the lack of site specificity of the UT-A1 phosphorylated at Ser-486 (Figure 9, B, D, and E, re- 32P labeling technique employed in the study, quantitative spectively). conclusions concerning phosphorylation of these sites could not be made. In our study, we show that phosphorylation at each of these two sites increased approximately 4-fold with DISCUSSION dDAVP exposure for 30 minutes. Because surrounding amino acid sequences from both sites are compatible with PKA as the The new advances in proteomic methodologies used here al- kinase involved,27 we performed in vitro assays that showed low an unbiased, large-scale, discovery-based approach for the PKA can phosphorylate both Ser-486 and Ser-499 (Figure 8). study of cell signaling. Here, we have applied these methodol- However, several other kinases have specificities similar to ogies to vasopressin signaling in the renal collecting duct. Be- PKA,29 and consequently the kinase responsible for in vivo cause water and urea transport are known to be vasopressin- phosphorylation of these sites cannot be specified with cer- regulated, we decided to emphasize our data focusing on tainty. phosphorylation of aquaporins and urea channels. However, a Our study also demonstrated a previously unknown site large-scale LC-MS/MS approach also allows unanticipated ob- (Ser-84) on the N-terminal tail of the urea channel that is phos- servations to be made regarding other parts of the signaling phorylated in response to dDAVP. Ser-84 is contained within a network. Here, we discuss examples of both types of observa- putative PKA motif,27 and an in vitro assay showed that PKA tions: (1) regulated phosphorylation of UT-A urea channel can phosphorylate this site (Figure 8). This site is present in isoforms and (2) regulation of ␤-catenin phosphorylation by both UT-A1 and UT-A3, and it is the only regulated site shared vasopressin. by both isoforms. The use of newly developed phosphospecific Our data identify and quantify multiple vasopressin-regu- UT-A antibodies definitively showed that phosphorylation of lated phosphorylation sites on the urea channel (UT-A1 and this site is indeed increased by dDAVP treatment in both iso- UT-A3). A schematic of these sites and their relative locations forms. on UT-A1 and UT-A3 is summarized in Figure 10. A previous Further evidence supporting a role for PKA in regulating study17 identified four phosphorylation sites (Ser-35, Ser-62, phosphorylation of collecting duct urea channels was found by Ser-63, and Ser-486). In this study, we confirm previously using the inhibitor H89, which nearly completely blocked identified sites and also report two previously unknown phos- phosphorylation at both Ser-84 and Ser-486. Results from phorylation sites (Ser-10 and Ser-84). Of the sites reported in U0126 inhibitor studies suggested that the MEK-ERK pathway this study, we demonstrate that three (Ser-84, Ser-486, and may also play a role in regulating phosphorylation of UT-A1 at Ser-499) are strongly regulated by vasopressin both by LC- Ser-84 but not at Ser-486. However, U0126 did not block the

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H89 U0126 9 vehicle µ µ (10 M) (10 M) - dDAVP A C 8 UT-A1 Ser(p)-84 + dDAVP - + - + - + dDAVP (1 nM) 7 kDa 6 *** 37 5 AQP2 Ser(p)-256 4 25 3

2

Normalized Abundance 1 ***

100 0 UT-A1 vehicle H89 (10 µM) U0126 (10 µM) Ser(p)-84 75 12 50 UT-A3 D UT-A3 Ser(p)-84 - dDAVP Ser(p)-84 10 + dDAVP 37 8

6

4 100 UT-A1 *** 2

Ser(p)-486 Normalized Abundance 75 0 vehicle H89 (10 µM) U0126 (10 µM)

7 100 total E - dDAVP UT-A1 Ser(p)-486 UT-A1 6 + dDAVP 75 5

50 4 total UT-A3 37 3 2

1 50 Abundance Normalized *** pERK 0 37 vehicle H89 (10 µM) U0126 (10 µM)

5 1.2 B 4.5 AQP2 Ser(p)-256 - dDAVP F pERK - dDAVP + dDAVP 1 + dDAVP 4

3.5 0.8 * 3 * 2.5 0.6 2 1.5 0.4

1 0.2 Normalized Abundance 0.5 Normalized Abundance *** *** 0 0 vehicle H89 (10 µM) U0126 (10 µM) vehicle H89 (10 µM) U0126 (10 µM)

Figure 9. Phosphorylation of both UT-A1 and UT-A3 is blocked by an inhibitor of PKA. (A) Rat IMCD samples were treated with either vehicle, the PKA inhibitor H89, or the MEK inhibitor U0126 for 10 minutes, followed by incubation with dDAVP for 20 minutes, followed by immunoblotting with antibodies to AQP2 Ser(p)-256, UT-A1 and UT-A3 Ser(p)-84, UT-A1 Ser(p)-486, total UT-A1 and UT-A3, and pERK. (B to F) Quantification of band densities from these immunoblots as well as other trials (n ϭ 3; *P Ͻ 0.05 versus vehicle; ***P Ͻ 0.001 versus vehicle). ability of dDAVP to increase phosphorylation of UT-A1 at charge (Ϫ1) as a phosphorylated serine and could conceivably either site or UT-A3 at Ser-84. Furthermore, neither Ser-84 nor represent a constitutively activated form. Ser-486 is part of a proline-directed consensus motif, which Other UT-A sites that were quantified but were shown to would be compatible with direct phosphorylation by a MAP not be regulated by vasopressin were Ser-10, Ser-62, and Ser- kinase.27 63. None of these sites have neighboring amino acids sugges- Regarding the evolutionary conservation of these sites, Ser- tive of a PKA or any other basophilic motif. However, these 499 is highly conserved across rat, mouse, horse, cow, and hu- sites may still be important for other regulatory pathways. man UT-A1. Ser-486 was also conserved in all of these species In addition to UT-A, our study successfully quantified except for cow, where it is an asparagine residue. Ser-84 was changes in site-specific phosphorylation of multiple other pro- less conserved, being replaced by aspartic acid in human and teins, including Ser-552 on ␤-catenin (Table 1 and Figure 5). arginine in horse. The aspartic acid in human has the same Ser-552 on ␤-catenin was found to undergo a large increase

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ulation. For example, septin-9 phosphorylation was strongly -499 Ser-10Ser-6Ser-842/63 Ser-486Ser down-regulated at Ser-67, a putative MAP kinase site. Septins are polymeric GTP-binding proteins that associate with cell UT-A1 H2N COOH 929 aa membranes as well as the actin and microtubule cytoskel- etons.38 Furthermore, a protein called ArfGAP with FG repeats 63 1 also undergoes a marked decrease in phosphorylation in re- Ser-10Ser-62/Ser-84 sponse to dDAVP at several proline-directed sites. This protein UT-A3 H2N COOH is believed to be involved in vesicle docking and fusion in ac- 460 aa rosome formation in spermatozoa39 and could play a role in Figure 10. Schematic of the two urea channels (UT-A1 and UT- regulation of membrane trafficking in the collecting duct. Fi- A3) expressed in IMCD with the relative position of phosphory- nally, epsin-3 underwent a marked decrease in phosphoryla- lation sites identified and quantified by LC-MS/MS. Sites marked tion at Thr-361 a putative MAP kinase site. This protein func- with an arrow were found to be phosphorylated in response to tions as a ubiquitin-dependent clathrin adaptor and may be vasopressin. involved in regulation of membrane trafficking in the collect- ing duct.40 in phosphorylation in response to vasopressin, both in vitro (Figure 5A) and in vivo (Figure 5B). ␤-Catenin is a well- studied cytoplasmic protein that has two general roles in CONCISE METHODS cells: (1) a structural role in cadherin-mediated cell adhe- sion and (2) a signaling role as a transcriptional coregula- IMCD Isolation tor.30 It is involved in the highly conserved canonical Wnt Sprague-Dawley rats were obtained between 1 and 3 months of age signaling pathway. Although involvement of ␤-catenin in (Animal Care and Use Committee protocol number H-0110). Furo- the embryonic development of the collecting duct has been semide was injected into the rats (intraperitoneally, 0.5 mg) 30 min- well studied,31–33 there is only indirect evidence regarding utes before euthanization. Both kidneys were removed, and the inner its relationship to vasopressin signaling in the mature col- medullas were dissected out. IMCD isolation was performed using a lecting duct.23 The amino acid sequence surrounding Ser- modified version of previously outlined methods.41,42 Inner medullas 552 of ␤-catenin (…QRRTS552MGGT…) is consistent with were minced with a razorblade and digested for approximately 75 a PKA motif.27 Through 32P labeling and site-directed mu- minutes at 37°C in an enzyme solution containing 3 mg/ml collage- tagenesis, it has been shown previously that PKA can phos- nase B and 3 mg/ml hyaluronidase dissolved in sucrose buffer (250 phorylate Ser-552.34 Subsequent studies demonstrated that mM sucrose and 10 mM triethanolamine, pH 7.6). After enzyme di- phosphorylation of Ser-552 by PKA can facilitate the inter- gestion, a low-speed spin (70 ϫ g, 20 seconds) was used to pellet action between ␤-catenin and T cell factor, a transcription IMCD cells. Cells were rinsed twice with sucrose buffer and once with factor that up-regulates gene expression related to cell bicarbonate buffer (118 mM NaCl, 25 mM NaHCO3, 5.5 mM glucose, 35 growth. Given the fact that we have shown vasopressin 5mMKCl,4mMNa2HPO4, 2 mM CaCl2, and 1.2 mM MgSO4,pH causes increased phosphorylation of Ser-552 on ␤-catenin 7.4). Cells were resuspended in bicarbonate buffer, and samples were in the collecting duct, an important future direction will be treated with either 1 nM dDAVP or vehicle for 30 minutes at 37°C in to elucidate whether similar interactions among vasopres- a pH- and temperature-controlled chamber. For studies using H89 sin, ␤-catenin, and expression of growth-promoting genes and U0126, the drug was added for a 10-minute preincubation period are also relevant for the collecting duct. followed by addition of 1 nM dDAVP for 20 minutes. Data in the Gene Ontology analysis and Conserved Domain analysis of phosphoproteome profiling experiment were derived only from a proteins identified in Table 1 did not point to any single cate- dDAVP-treated sample without a vehicle-treated control (Figure 1A). gory of proteins that are regulated by dDAVP (Supplementary Data). However, several proteins whose phosphorylation is in- Reduction, Alkylation, and In-Solution Protease creased have potential roles in regulation of aquaporin-2 Digestion and/or UT-A isoforms. For example, the PCTAIRE-3 protein After dDAVP treatment, IMCD tubules were resuspended in lysis kinase was strongly phosphorylated at Ser-66, a putative PKA buffer (8 M urea, 75 mM NaCl, 50 mM Tris-HCl, with protease and site, in response to dDAVP. This kinase is differentially ex- phosphatase inhibitors). Cells were sonicated with 0.5-second pulses pressed in IMCD versus non-collecting duct cells3 and is for 30 cycles followed by centrifugation at 10,000 ϫ g for 10 minutes. thought to be involved in membrane trafficking.36 In addition, Protein concentration in the supernatants was determined using the plexin domain containing-2 is also strongly phosphorylated at BCA protein assay (Pierce, Rockford, IL). Approximately 1 mg of a putative PKA site (Ser-507). This protein has been shown to protein was used for large-scale phosphoproteome profiling, whereas bind cortactin, which is an important regulator of actin poly- 500 ␮g was used for quantitative studies. Samples were reduced for 1 merization at cell adhesion sites.37 In contrast, several phos- hour with 10 mM dithiothreitol, followed by alkylation with 40 mM phorylation sites were found to be strongly down-regulated in iodoacetamide for 1 hour in the dark. 40 mM dithiothreitol was added proteins that have relevance to aquaporin-2 and/or UT-A reg- to quench unreacted iodoacetamide. Samples were diluted to a urea

312 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 303–315, 2010 www.jasn.org BASIC RESEARCH concentration of Ͻ1 M and digested with trypsin (Promega, Madi- 50% solvent B for 5 minutes; and 50% to 95% solvent B for 5 minutes son, WI) overnight at 37°C in 50 mM ammonium bicarbonate. For (solvent A: 0.1% formic acid in 98% water, 2% ACN; solvent B: 0.1% samples digested with chymotrypsin (Roche, Penzberg, Germany), formic acid in 100% ACN). LTQ-Orbitrap settings were as follows: the reaction was performed for 16 hours at 25°C in 100 mM Tris-HCl spray voltage 1.5 kV; full MS mass range: m/z 300 to 2000; and MS/MS and 5 mM CaCl2. mass range: m/z 100 to 2000. The LTQ-Orbitrap was operated in a data-dependent mode; i.e., one MS1 high resolution scan for precur- Desalting, Phosphopeptide Enrichment, and Sample sor ions followed by three data-dependent MS2 scans for precursor Fractionation ions above a threshold ion count of 2000 with collision energy of 35%. After protease digestion, peptide samples were desalted using a 1-cc In some cases, parent ion masses were entered into an inclusion list for HLB cartridge (Waters, Milford, MA) and dried by SpeedVac. The TIS experiments. MS3 scans were automatically triggered on the basis dried peptides were resuspended in 300 ␮l of solvent A (5 mM of the observation of neutral loss peaks of phosphoric acids from

KH2PO4, 25% acetonitrile (ACN), pH 2.7) and injected onto a Poly- precursor ions in MS2 scans that met a threshold criterion of 500 Sulfoethyl A SCX column (4.6-mm inner diameter ϫ 20-cm length, counts. 5-␮m particle size, 300-Å pore size). SCX chromatography was car- ried out on an Agilent HP1100 system at 1 ml/min flow rate using the ETD and Decision Tree-Driven Tandem MS/MS following gradient: 0% solvent B for 2 minutes; 0 to 20% solvent B for ETD parameters were tuned using bovine growth hormone-releasing 40 minutes; 20% to 100% solvent B for 5 minutes; and 100% solvent receptor (Sigma G0644). The reaction time was optimized at 85 mil-

B held for 5 minutes (solvent B: 5 mM KH2PO4, 25% ACN, and 350 liseconds (with an AGC target for fluoranthene radical anion of mM KCl, pH 2.7). UV absorbance at 214 nm was monitored while 300,000). Online mass spectrometric experiments were performed by fractions were collected at 1.5-ml intervals and dried down by Speed- MS1 analysis in the Orbitrap at 30,000 resolution followed by five Vac. All 24 fractions were desalted on 1-cc HLB cartridges (Waters) data-dependent MS/MS events with product ion analysis performed before phosphopeptide enrichment. SCX was not used in other anal- in the LTQ. The precursor ions selected for MS/MS were determined ysis methods, such as TIS or label-free LC-MS/MS quantitation. by intensity within MS1 (Ն1000 counts) followed by dynamic exclu- Phosphopeptide enrichment was performed as described17 using im- sion (30 seconds or a peak list of 500) and charge state inclusion (ions mobilized metal affinity chromatography (IMAC) with a Ga3ϩ matrix with two or more charges). The form of MS/MS activation was either (Phosphopeptide Isolation Kit; Pierce). Before analysis by MS/MS, ETD-only, CID-only, or decision tree-based selection.43 When using samples were desalted using C18 Ziptips (Millipore) and resuspended the decision tree-based selection, the dissociation method for every in 0.1% formic acid. MS/MS event was determined in real time in an automated fashion on the basis of the precursor z and m/z. This feature was fully incorpo- LC-MS/MS Analysis on Linear Trap Quadrupole rated into the LTQ Tune 2.5.5 instrument control software (default An Agilent 1100 nanoflow LC system (Palo Alto, CA) connected to a setting). Finnigan linear trap quadrupole (LTQ) mass spectrometer (Thermo Electron, San Jose, CA) was used for LC-MS/MS analysis. Peptide Data Searching, Scoring, Quantification, and trapping was performed at a flow rate of 2 ␮l/min. Peptides were Bioinformatics eluted onto a reversed-phase PicoFrit column (New Objective, Searches were performed using the latest version of the rat RefSeq Woburn, MA), and separation was performed using a linear ACN database (National Center for Biotechnology Information), with con- gradient (0 to 60%) in 0.1% formic acid. Peptides were introduced catenated forward and reversed sequences to allow for target-decoy into the LTQ mass spectrometer using a nanospray ion source. The analysis. The database also contained sequences for common MS con- spray voltage was set to 2 kV and collision energy to 35%. Data- taminants, such as human keratin and porcine trypsin. Data from the dependent acquisition mode (dynamic exclusion enabled) was used phosphoproteome profiling experiment were searched with SE- for profiling experiments. Survey MS1 scans were followed by five QUEST,44 InsPecT,45,46 and OMSSA.47 InsPecT and OMSSA searches MS2 scans. TIS was performed with predetermined masses entered used the high-performance computational capabilities of the Biowulf into an inclusion list (dynamic exclusion disabled). Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov). All of the other data were searched using LC-MS/MS Analysis on LTQ-Orbitrap SEQUEST only. Filtering and determination of false-discovery rate All dried fractions were analyzed on an LTQ-Orbitrap (Thermo- were done using methods described previously.48,49 All datasets were Fisher Scientific LLC) interfaced with an Eksigent nano-LC 1D plus filtered for a false-discovery rate of Ͻ2%. Phosphorylation site local- system (Eksigent Technologies LLC, Dublin, CA) using CID fragmen- ization was performed using Ascore50 and PhosphoScore.51 Label-free tation. Briefly, samples were loaded onto an Agilent Zorbax 300SB- phosphopeptide quantification was performed using QUOIL.22 Gene C18 trap column (0.3-mm inner diameter ϫ 5-mm length, 5-␮m Ontology and Conserved Domain Database analyses were performed particle size) at a flow rate of 5 ␮l/min for 10 minutes. Reversed-phase using in-house software.

C18 chromatographic separation of peptides was carried out on a pre- packed PicoFrit column (75-␮m inner diameter ϫ 10-cm length; Brattleboro Rats New Objective) at 300 nl/min using the following gradient: 2% to 5% Brattleboro rats (Animal Care and Use Committee protocol number solvent B for 5 minutes; 5% to 45% solvent B for 60 minutes; 45% to H-0110) were injected with dDAVP (2 nmol dissolved in saline) or

J Am Soc Nephrol 21: 303–315, 2010 Phosphoproteomics of IMCD 313 BASIC RESEARCH www.jasn.org vehicle intramuscularly and then euthanized after 45 minutes. Inner 2. Brown D: The ins and outs of aquaporin-2 trafficking. Am J Physiol medullas were dissected, homogenized, and solubilized in Laemmli Renal Physiol 284: F893–F901, 2003 3. Uawithya P, Pisitkun T, Ruttenberg BE, Knepper MA: Transcriptional buffer as described previously.9 profiling of native inner medullary collecting duct cells from rat kidney. Physiol Genomics 32: 229–253, 2008 Antibodies 4. Yano Y, Rodrigues AC, Jr., de Braganca AC, Andrade LC, Magaldi AJ: Affinity-purified rabbit polyclonal antibodies against rat Ser(p)-256- PKC stimulated by glucagon decreases UT-A1 urea transporter ex- AQP252 and total UT-A1 (L448)24 have been previously described. pression in rat IMCD. Pflugers Arch 456: 1229–1237, 2008 Phosphospecific antibodies were generated against sequences sur- 5. O’Connor PM, Cowley AW, Jr.: Vasopressin-induced nitric oxide pro- duction in rat inner medullary collecting duct is dependent on V2 rounding residues Ser-84 and Ser-486 of rat UT-A1 (PhosphoSolu- receptor activation of the phosphoinositide pathway. Am J Physiol tions, Aurora, CO). Specificity of these antibodies was established by Renal Physiol 293: F526–F532, 2007 dotblot (data not shown) and immunoblot. A phosphospecific anti- 6. Umenishi F, Narikiyo T, Vandewalle A, Schrier RW: cAMP regulates body recognizing Ser(p)-552-␤-catenin was obtained from Cell Sig- vasopressin-induced AQP2 expression via protein kinase A-indepen- naling Technology (product no. 9566; Danvers, MA). dent pathway. Biochim Biophys Acta 1758: 1100–1105, 2006 7. Mordasini D, Bustamante M, Rousselot M, Martin PY, Hasler U, Feraille E: Stimulation of Naϩ transport by AVP is independent of PKA phos- Immunoblot Analysis phorylation of the Na-K-ATPase in collecting duct principal cells. Am J Immunoblotting was used to assess the response of AQP2 and UT-A1 Physiol Renal Physiol 289: F1031–F1039, 2005 to dDAVP treatment and was carried out using previously described 8. Tamma G, Klussmann E, Maric K, Aktories K, Svelto M, Rosenthal W, methods.17,48 Valenti G: Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells. Am J Physiol Renal Physiol 281: F1092–F1101, 2001 In vitro Phosphorylation Assay 9. Pisitkun T, Jacob V, Schleicher SM, Chou CL, Yu MJ, Knepper MA: Akt We performed an in vitro assay to determine whether particular serine and ERK1/2 pathways are components of the vasopressin signaling residues contained within in silico-predicted PKA phosphorylation network in rat native IMCD. Am J Physiol Renal Physiol 295: F1030– consensus motifs in UT-A1 could be phosphorylated by PKA. We F1043, 2008 obtained synthetic peptides corresponding to sequences surrounding 10. Chou CL, Christensen BM, Frische S, Vorum H, Desai RA, Hoffert JD, the S84, S486, and S499 residues of UT-A1 (AnaSpec, San Jose, CA). de Lanerolle P, Nielsen S, Knepper MA: Non-muscle myosin II and myosin light chain kinase are downstream targets for vasopressin We used a peptide with the amino acid sequence of the C-terminal tail signaling in the renal collecting duct. J Biol Chem 279: 49026–49035, of AQP218 as a positive control. We incubated a total of 5 ␮g of each 2004 peptide with 1ϫ kinase buffer and 200 ␮M ATP (Cell Signaling Tech- 11. Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper nology, Beverly, MA) in the presence or absence of PKA C-␣ kinase MA: Vasopressin increases water permeability of kidney collecting (Cell Signaling Technology, Beverly, MA) for 1 hour at 30°C. After duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad SciUSA92: 1013–1017, 1995 incubation, the reaction mixtures were digested overnight with either 12. Sands JM, Nonoguchi H, Knepper MA: Vasopressin effects on urea trypsin at 37°C or chymotrypsin (for Ser-84 peptide) at 25°C. Samples and H2O transport in inner medullary collecting duct subsegments. were then desalted, resuspended in 0.1% formic acid, and analyzed on Am J Physiol 253: F823–F832, 1987 ϩ an LTQ mass spectrometer (Thermo Electron). 13. Snyder PM: Minireview: Regulation of epithelial Na channel traffick- ing. Endocrinology 146: 5079–5085, 2005 14. Tomita K, Pisano JJ, Knepper MA: Control of sodium and potassium transport in the cortical collecting duct of the rat. Effects of bradykinin, ACKNOWLEDGMENTS vasopressin, and deoxycorticosterone. J Clin Invest 76: 132–136, 1985 15. DiGiovanni SR, Nielsen S, Christensen EI, Knepper MA: Regulation of This work was funded by the intramural budget of the National Heart, collecting duct water channel expression by vasopressin in Brattleboro rat. Proc Natl Acad SciUSA91: 8984–8988, 1994 Lung, and Blood Institute (Z01-HL-01285-KE). A.D.B. is supported 16. Terris J, Ecelbarger CA, Nielsen S, Knepper MA: Long-term regulation by the Howard Hughes Medical Institute-National Institutes of of four renal aquaporins in rats. Am J Physiol 271: F414–F422, 1996 Health Research Scholars Program and the New York University 17. Hoffert JD, Pisitkun T, Wang G, Shen RF, Knepper MA: Quantitative School of Medicine. We would like to thank Guozhong Ma, Center for phosphoproteomics of vasopressin-sensitive renal cells: Regulation of Biomedical Informatics, National Heart, Lung, and Blood Institute, aquaporin-2 phosphorylation at two sites. Proc Natl Acad SciUSA 103: 7159–7164, 2006 National Institutes of Health, for his assistance with the Web publish- 18. Hoffert JD, Fenton RA, Moeller HB, Simons B, Tchapyjnikov D, McDill ing of the Collecting Duct Phosphoproteome Database. BW, Yu MJ, Pistikun T, Chen F, Knepper MA: Vasopressin-stimulated increase in phosphorylation at ser-269 potentiates plasma membrane retention of aquaporin-2. J Biol Chem 283: 24617–24627, 2008 DISCLOSURES 19. Blount MA, Klein JD, Martin CF, Tchapyjnikov D, Sands JM: Forskolin None. stimulates phosphorylation and membrane accumulation of UT-A3. Am J Physiol Renal Physiol 293: F1308–F1313, 2007 20. Blount MA, Mistry AC, Frohlich O, Price SR, Chen G, Sands JM, Klein REFERENCES JD: Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accu- 1. Nielsen S, Frokiaer J, Marples D, Kwon TH, Agre P, Knepper MA: mulation. 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314 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 303–315, 2010 www.jasn.org BASIC RESEARCH

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J Am Soc Nephrol 21: 303–315, 2010 Phosphoproteomics of IMCD 315 Input Gene Symbol GO category GO Term NP_001019962 Epn3 component cell cortex NP_001019962 Epn3 component clathrin-coated vesicle NP_001019962 Epn3 component cytoplasm NP_001019962 Epn3 component cytoplasmic vesicle NP_001019962 Epn3 component nucleus NP_001019962 Epn3 component perinuclear region of cytoplasm NP_001019962 Epn3 function lipid binding NP_001019962 Epn3 process not classified NP_001101454 Pdik1l component not classified NP_001101454 Pdik1l function not classified NP_001101454 Pdik1l process not classified NP_001124461 Ptges3 component cellular_component NP_001124461 Ptges3 component cytoplasm NP_001124461 Ptges3 function isomerase activity NP_001124461 Ptges3 function prostaglandin-E synthase activity NP_001124461 Ptges3 function unfolded protein binding NP_001124461 Ptges3 process fatty acid biosynthetic process NP_001124461 Ptges3 process prostaglandin biosynthetic process NP_001129068 Agfg1 component cytoplasmic membrane-bounded vesicle NP_001129068 Agfg1 component nucleus NP_001129068 Agfg1 function ARF GTPase activator activity NP_001129068 Agfg1 function DNA binding NP_001129068 Agfg1 function metal ion binding NP_001129068 Agfg1 function zinc ion binding NP_001129068 Agfg1 process cell differentiation NP_001129068 Agfg1 process multicellular organismal development NP_001129068 Agfg1 process regulation of ARF GTPase activity NP_001129068 Agfg1 process spermatogenesis NP_001129068 Agfg1 process transport NP_037041 Aqp2 component apical plasma membrane NP_037041 Aqp2 component apical plasma membrane NP_037041 Aqp2 component apical plasma membrane NP_037041 Aqp2 component basolateral plasma membrane NP_037041 Aqp2 component cell fraction NP_037041 Aqp2 component clathrin-coated vesicle NP_037041 Aqp2 component cytoplasm NP_037041 Aqp2 component early endosome NP_037041 Aqp2 component exocytic vesicle NP_037041 Aqp2 component integral to membrane NP_037041 Aqp2 component lysosome NP_037041 Aqp2 component membrane fraction NP_037041 Aqp2 component membrane fraction NP_037041 Aqp2 component plasma membrane NP_037041 Aqp2 component plasma membrane NP_037041 Aqp2 component protein complex NP_037041 Aqp2 component recycling endosome NP_037041 Aqp2 component recycling endosome NP_037041 Aqp2 component rough endoplasmic reticulum NP_037041 Aqp2 component trans-Golgi network NP_037041 Aqp2 function actin binding NP_037041 Aqp2 function protein binding NP_037041 Aqp2 function transporter activity NP_037041 Aqp2 function water channel activity NP_037041 Aqp2 function water channel activity NP_037041 Aqp2 function water channel activity NP_037041 Aqp2 process actin filament depolymerization NP_037041 Aqp2 process aging NP_037041 Aqp2 process apoptosis NP_037041 Aqp2 process cell volume homeostasis NP_037041 Aqp2 process cellular response to water deprivation NP_037041 Aqp2 process cellular response to water deprivation NP_037041 Aqp2 process female pregnancy NP_037041 Aqp2 process hyperosmotic response NP_037041 Aqp2 process positive regulation of calcium ion transport NP_037041 Aqp2 process renal water transport NP_037041 Aqp2 process renal water transport NP_037041 Aqp2 process response to calcium ion NP_037041 Aqp2 process response to glucagon stimulus NP_037041 Aqp2 process response to hormone stimulus NP_037041 Aqp2 process response to lipopolysaccharide NP_037041 Aqp2 process response to lithium ion NP_037041 Aqp2 process response to salt stress NP_037041 Aqp2 process response to starvation NP_037041 Aqp2 process response to water deprivation NP_037041 Aqp2 process transport NP_037041 Aqp2 process water homeostasis NP_037041 Aqp2 process water transport NP_037041 Aqp2 process water transport NP_037041 Aqp2 process water transport NP_110484 Lyn component Golgi apparatus NP_110484 Lyn component alpha2-beta1 integrin complex NP_110484 Lyn component cell-cell adherens junction NP_110484 Lyn component cytoplasm NP_110484 Lyn component cytosol NP_110484 Lyn component membrane fraction NP_110484 Lyn component membrane raft NP_110484 Lyn component membrane raft NP_110484 Lyn component mitochondrial crista NP_110484 Lyn component mitochondrial intermembrane space NP_110484 Lyn component mitochondrial membrane NP_110484 Lyn component nucleus NP_110484 Lyn component perinuclear region of cytoplasm NP_110484 Lyn component plasma membrane NP_110484 Lyn component plasma membrane NP_110484 Lyn component postsynaptic density NP_110484 Lyn function SH3 domain binding NP_110484 Lyn function enzyme binding NP_110484 Lyn function gamma-tubulin binding NP_110484 Lyn function glycosphingolipid binding NP_110484 Lyn function integrin binding NP_110484 Lyn function kinase activity NP_110484 Lyn function phosphoprotein binding NP_110484 Lyn function platelet-derived growth factor receptor binding NP_110484 Lyn function protein binding NP_110484 Lyn function protein binding NP_110484 Lyn function protein complex binding NP_110484 Lyn function protein complex binding NP_110484 Lyn function protein kinase activity NP_110484 Lyn function protein tyrosine kinase activity NP_110484 Lyn function protein tyrosine kinase activity NP_110484 Lyn function protein tyrosine kinase activity NP_110484 Lyn function receptor binding NP_110484 Lyn function ubiquitin protein ligase binding NP_110484 Lyn process B cell receptor signaling pathway NP_110484 Lyn process B cell receptor signaling pathway NP_110484 Lyn process cellular response to extracellular stimulus NP_110484 Lyn process cellular response to heat NP_110484 Lyn process cytokine secretion NP_110484 Lyn process erythrocyte differentiation NP_110484 Lyn process histamine secretion by mast cell NP_110484 Lyn process intracellular signaling cascade NP_110484 Lyn process intracellular signaling cascade NP_110484 Lyn process oligodendrocyte development NP_110484 Lyn process peptidyl-tyrosine phosphorylation NP_110484 Lyn process peptidyl-tyrosine phosphorylation NP_110484 Lyn process positive regulation of Fc receptor mediated stimulatory signaling pathway NP_110484 Lyn process positive regulation of cell proliferation NP_110484 Lyn process positive regulation of glial cell proliferation NP_110484 Lyn process positive regulation of oligodendrocyte progenitor proliferation NP_110484 Lyn process positive regulation of phosphorylation NP_110484 Lyn process positive regulation of tyrosine phosphorylation of STAT protein NP_110484 Lyn process protein amino acid autophosphorylation NP_110484 Lyn process regulation of release of sequestered calcium ion into cytosol NP_110484 Lyn process response to amino acid stimulus NP_110484 Lyn process response to axon injury NP_110484 Lyn process response to carbohydrate stimulus NP_110484 Lyn process response to drug NP_110484 Lyn process response to hormone stimulus NP_110484 Lyn process response to insulin stimulus NP_110484 Lyn process response to organic cyclic substance NP_110484 Lyn process response to peptide hormone stimulus NP_110484 Lyn process response to protein stimulus NP_110484 Lyn process response to sterol depletion NP_110484 Lyn process response to toxin NP_445747 Cast component not classified NP_445747 Cast function protein binding NP_445747 Cast process myoblast fusion NP_445747 Cast process negative regulation of catalytic activity NP_445747 Cast process regulation of protein catabolic process NP_446225 Tjp2 component cytoplasm NP_446225 Tjp2 component gap junction NP_446225 Tjp2 component plasma membrane NP_446225 Tjp2 component plasma membrane NP_446225 Tjp2 component tight junction NP_446225 Tjp2 component tight junction NP_446225 Tjp2 function protein C-terminus binding NP_446225 Tjp2 function protein binding NP_446225 Tjp2 process response to organic substance NP_742005 Canx component axon NP_742005 Canx component cell soma NP_742005 Canx component cytoplasm NP_742005 Canx component dendrite cytoplasm NP_742005 Canx component dendritic spine NP_742005 Canx component endoplasmic reticulum NP_742005 Canx component endoplasmic reticulum NP_742005 Canx component endoplasmic reticulum NP_742005 Canx component integral to membrane NP_742005 Canx component integral to membrane NP_742005 Canx component melanosome NP_742005 Canx component membrane NP_742005 Canx component ribosome NP_742005 Canx component rough endoplasmic reticulum NP_742005 Canx function calcium ion binding NP_742005 Canx function protein binding NP_742005 Canx function protein binding NP_742005 Canx function protein binding NP_742005 Canx function sugar binding NP_742005 Canx function unfolded protein binding NP_742005 Canx process aging NP_742005 Canx process protein folding NP_786937 Hsp90aa1 component cytoplasm NP_786937 Hsp90aa1 component cytoplasm NP_786937 Hsp90aa1 component cytosol NP_786937 Hsp90aa1 component intracellular NP_786937 Hsp90aa1 component intracellular NP_786937 Hsp90aa1 function ATP binding NP_786937 Hsp90aa1 function ATP binding NP_786937 Hsp90aa1 function TPR domain binding NP_786937 Hsp90aa1 function nitric-oxide synthase regulator activity NP_786937 Hsp90aa1 function nitric-oxide synthase regulator activity NP_786937 Hsp90aa1 function protein binding NP_786937 Hsp90aa1 function protein binding NP_786937 Hsp90aa1 function protein homodimerization activity NP_786937 Hsp90aa1 function protein homodimerization activity NP_786937 Hsp90aa1 function unfolded protein binding NP_786937 Hsp90aa1 function unfolded protein binding NP_786937 Hsp90aa1 process cellular chaperone-mediated protein complex assembly NP_786937 Hsp90aa1 process mitochondrial outer membrane translocase complex assembly NP_786937 Hsp90aa1 process positive regulation of nitric oxide biosynthetic process NP_786937 Hsp90aa1 process positive regulation of nitric oxide biosynthetic process NP_786937 Hsp90aa1 process protein folding NP_786937 Hsp90aa1 process protein folding NP_786937 Hsp90aa1 process response to stress NP_786941 Psip1 component cytosol NP_786941 Psip1 function not classified NP_786941 Psip1 process initiation of viral infection NP_786941 Psip1 process provirus integration NP_789826 Sept9 component cellular_component NP_789826 Sept9 component microtubule NP_789826 Sept9 component perinuclear region of cytoplasm NP_789826 Sept9 component stress fiber NP_789826 Sept9 function ATP binding NP_789826 Sept9 function DNA binding NP_789826 Sept9 function DNA topoisomerase (ATP-hydrolyzing) activity NP_789826 Sept9 function GTP binding NP_789826 Sept9 function molecular_function NP_789826 Sept9 function protein binding NP_789826 Sept9 process DNA topological change NP_789826 Sept9 process biological_process NP_789826 Sept9 process cell cycle NP_789826 Sept9 process protein heterooligomerization NP_808877 Slc14a2 component integral to membrane NP_808877 Slc14a2 function copper ion binding NP_808877 Slc14a2 function protein binding NP_808877 Slc14a2 function urea transmembrane transporter activity NP_808877 Slc14a2 function urea transmembrane transporter activity NP_808877 Slc14a2 function urea transmembrane transporter activity NP_808877 Slc14a2 process oxidation reduction NP_808877 Slc14a2 process response to water deprivation NP_808877 Slc14a2 process urea transport NP_808877 Slc14a2 process urea transport NP_835196 Abca1 component Golgi apparatus NP_835196 Abca1 component Golgi apparatus NP_835196 Abca1 component caveola NP_835196 Abca1 component integral to plasma membrane NP_835196 Abca1 component integral to plasma membrane NP_835196 Abca1 component intracellular membrane-bounded organelle NP_835196 Abca1 component membrane fraction NP_835196 Abca1 component membrane raft NP_835196 Abca1 component phagocytic vesicle NP_835196 Abca1 component plasma membrane NP_835196 Abca1 component plasma membrane NP_835196 Abca1 component plasma membrane NP_835196 Abca1 function ATP binding NP_835196 Abca1 function ATPase activity NP_835196 Abca1 function ATPase activity, coupled to transmembrane movement of substances NP_835196 Abca1 function ATPase activity, coupled to transmembrane movement of substances NP_835196 Abca1 function anion transmembrane transporter activity NP_835196 Abca1 function anion transmembrane transporter activity NP_835196 Abca1 function apolipoprotein A-I receptor activity NP_835196 Abca1 function cholesterol transporter activity NP_835196 Abca1 function cholesterol transporter activity NP_835196 Abca1 function high-density lipoprotein binding NP_835196 Abca1 function high-density lipoprotein receptor binding NP_835196 Abca1 function nucleotide binding NP_835196 Abca1 function phospholipid transporter activity NP_835196 Abca1 function phospholipid transporter activity NP_835196 Abca1 function protein binding NP_835196 Abca1 function protein binding NP_835196 Abca1 function protein binding NP_835196 Abca1 function protein binding NP_835196 Abca1 function small GTPase binding NP_835196 Abca1 function syntaxin-13 binding NP_835196 Abca1 process Cdc42 protein signal transduction NP_835196 Abca1 process G-protein coupled receptor protein signaling pathway NP_835196 Abca1 process cholesterol efflux NP_835196 Abca1 process cholesterol efflux NP_835196 Abca1 process cholesterol homeostasis NP_835196 Abca1 process cholesterol metabolic process NP_835196 Abca1 process cholesterol metabolic process NP_835196 Abca1 process cholesterol transport NP_835196 Abca1 process cholesterol transport NP_835196 Abca1 process cholesterol transport NP_835196 Abca1 process endosome transport NP_835196 Abca1 process high-density lipoprotein particle assembly NP_835196 Abca1 process interleukin-1 beta secretion NP_835196 Abca1 process intracellular cholesterol transport NP_835196 Abca1 process lipoprotein biosynthetic process NP_835196 Abca1 process lipoprotein biosynthetic process NP_835196 Abca1 process lipoprotein metabolic process NP_835196 Abca1 process lysosome organization NP_835196 Abca1 process peptide secretion NP_835196 Abca1 process peptide secretion NP_835196 Abca1 process phagocytosis, engulfment NP_835196 Abca1 process phagocytosis, engulfment NP_835196 Abca1 process phospholipid efflux NP_835196 Abca1 process phospholipid efflux NP_835196 Abca1 process phospholipid homeostasis NP_835196 Abca1 process phospholipid translocation NP_835196 Abca1 process phospholipid translocation NP_835196 Abca1 process phospholipid transport NP_835196 Abca1 process phospholipid transport NP_835196 Abca1 process platelet dense granule organization NP_835196 Abca1 process positive regulation of cAMP biosynthetic process NP_835196 Abca1 process protein amino acid lipidation NP_835196 Abca1 process protein amino acid lipidation NP_835196 Abca1 process response to drug NP_835196 Abca1 process response to nutrient NP_835196 Abca1 process reverse cholesterol transport NP_835196 Abca1 process reverse cholesterol transport XP_001053711 Nxf3 component cytoplasm XP_001053711 Nxf3 component intracellular XP_001053711 Nxf3 component nuclear RNA export factor complex XP_001053711 Nxf3 component nucleus XP_001053711 Nxf3 component nucleus XP_001053711 Nxf3 function mRNA binding XP_001053711 Nxf3 function nucleotide binding XP_001053711 Nxf3 function protein binding XP_001053711 Nxf3 process mRNA transport XP_001053711 Nxf3 process poly(A)+ mRNA export from nucleus XP_001053711 Nxf3 process transport XP_001059640 LOC498453 component nucleus XP_001059640 LOC498453 function DNA binding XP_001059640 LOC498453 function RNA polymerase II transcription factor activity XP_001059640 LOC498453 function protein binding XP_001059640 LOC498453 function transcription elongation regulator activity XP_001059640 LOC498453 function translation elongation factor activity XP_001059640 LOC498453 function zinc ion binding XP_001059640 LOC498453 process RNA elongation XP_001059640 LOC498453 process regulation of transcription from RNA polymerase II promoter XP_001071043 Sntb2 component synapse XP_001071043 Sntb2 function protein binding XP_001071043 Sntb2 process not classified XP_001078032 Ahnak component not classified XP_001078032 Ahnak function protein binding XP_001078032 Ahnak process not classified XP_237286 Tns1 component not classified XP_237286 Tns1 function protein binding XP_237286 Tns1 process intracellular signaling cascade XP_341257 RGD1564174 component not classified XP_341257 RGD1564174 function protein binding XP_341257 RGD1564174 process intracellular signaling cascade

Statistic: component cytoplasm 9 plasma membrane 9 nucleus 6 not classified 5 integral to membran 4 membrane fraction 4 perinuclear region of 3 apical plasma memb 3 Golgi apparatus 3 intracellular 3 cytosol 3 membrane raft 3 endoplasmic reticulu 3 clathrin-coated vesic 2 cellular_component 2 integral to plasma m 2 recycling endosome 2 rough endoplasmic r 2 tight junction 2 cell cortex 1 postsynaptic density 1 mitochondrial memb 1 mitochondrial interm 1 mitochondrial crista 1 melanosome 1 membrane 1 cell-cell adherens ju 1 alpha2-beta1 integri 1 ribosome 1 trans-Golgi network 1 gap junction 1 axon 1 protein complex 1 nuclear RNA export 1 caveola 1 lysosome 1 cell soma 1 exocytic vesicle 1 early endosome 1 cell fraction 1 basolateral plasma m 1 microtubule 1 cytoplasmic membra 1 dendrite cytoplasm 1 intracellular membra 1 stress fiber 1 phagocytic vesicle 1 cytoplasmic vesicle 1 synapse 1 dendritic spine 1 Total = 101

Statistic: function protein binding 22 unfolded protein bind 4 ATP binding 4 DNA binding 3 water channel activit 3 protein tyrosine kina 3 urea transmembrane 3 not classified 2 nucleotide binding 2 zinc ion binding 2 protein complex bind 2 cholesterol transport 2 nitric-oxide synthase 2 anion transmembran 2 ATPase activity, cou 2 protein homodimeriz 2 phospholipid transpo 2 lipid binding 1 molecular_function 1 GTP binding 1 DNA topoisomerase 1 copper ion binding 1 ATPase activity 1 TPR domain binding 1 RNA polymerase II t 1 sugar binding 1 calcium ion binding 1 protein C-terminus b 1 ubiquitin protein liga 1 receptor binding 1 small GTPase bindin 1 protein kinase activit 1 apolipoprotein A-I re 1 platelet-derived grow 1 phosphoprotein bind 1 kinase activity 1 integrin binding 1 glycosphingolipid bin 1 gamma-tubulin bindi 1 enzyme binding 1 SH3 domain binding 1 syntaxin-13 binding 1 transporter activity 1 translation elongatio 1 actin binding 1 high-density lipoprot 1 metal ion binding 1 mRNA binding 1 ARF GTPase activat 1 transcription elongat 1 prostaglandin-E synt 1 isomerase activity 1 high-density lipoprot 1 Total = 98

Statistic: process not classified 4 intracellular signaling 4 transport 3 water transport 3 protein folding 3 cholesterol transport 3 aging 2 protein amino acid li 2 cellular response to 2 renal water transpor 2 phospholipid transpo 2 phospholipid translo 2 response to hormon 2 phospholipid efflux 2 phagocytosis, engulf 2 peptide secretion 2 response to water de 2 B cell receptor signa 2 lipoprotein biosynthe 2 peptidyl-tyrosine pho 2 response to drug 2 reverse cholesterol t 2 positive regulation o 2 urea transport 2 cholesterol metaboli 2 cholesterol efflux 2 regulation of transcr 1 G-protein coupled re 1 Cdc42 protein signa 1 cholesterol homeost 1 oxidation reduction 1 protein heterooligom 1 cell cycle 1 biological_process 1 DNA topological cha 1 provirus integration 1 initiation of viral infec 1 response to stress 1 response to nutrient 1 mitochondrial outer m 1 cellular chaperone-m 1 endosome transport 1 response to organic 1 regulation of protein 1 negative regulation o 1 myoblast fusion 1 response to toxin 1 response to sterol de 1 response to protein 1 response to peptide 1 response to organic 1 response to insulin s 1 high-density lipoprot 1 response to carbohy 1 response to axon inj 1 response to amino a 1 regulation of release 1 protein amino acid a 1 positive regulation o 1 positive regulation o 1 positive regulation o 1 positive regulation o 1 positive regulation o 1 positive regulation o 1 interleukin-1 beta se 1 oligodendrocyte dev 1 RNA elongation 1 histamine secretion 1 erythrocyte different 1 cytokine secretion 1 cellular response to 1 cellular response to 1 intracellular choleste 1 mRNA transport 1 water homeostasis 1 lipoprotein metabolic 1 response to starvatio 1 response to salt stre 1 response to lithium i 1 response to lipopoly 1 lysosome organizati 1 response to glucago 1 response to calcium 1 phospholipid homeo 1 positive regulation o 1 hyperosmotic respon 1 female pregnancy 1 platelet dense granu 1 cell volume homeost 1 apoptosis 1 positive regulation o 1 actin filament depoly 1 poly(A)+ mRNA exp 1 spermatogenesis 1 regulation of ARF G 1 multicellular organis 1 cell differentiation 1 prostaglandin biosyn 1 fatty acid biosynthet 1 Total = 133 Input Gene Symbol GO category GO Term NP_001019932 Lrrfip2 component not classified NP_001019932 Lrrfip2 function not classified NP_001019932 Lrrfip2 process Wnt receptor signaling pathway NP_001093976 Pctk3 component not classified NP_001093976 Pctk3 function ATP binding NP_001093976 Pctk3 function cyclin-dependent protein kinase activity NP_001093976 Pctk3 function nucleotide binding NP_001093976 Pctk3 function protein binding NP_001093976 Pctk3 function transferase activity NP_001093976 Pctk3 process protein amino acid phosphorylation NP_001101892 Plxdc2 component membrane NP_001101892 Plxdc2 function not classified NP_001101892 Plxdc2 process not classified NP_037041 Aqp2 component apical plasma membrane NP_037041 Aqp2 component apical plasma membrane NP_037041 Aqp2 component apical plasma membrane NP_037041 Aqp2 component basolateral plasma membrane NP_037041 Aqp2 component cell fraction NP_037041 Aqp2 component clathrin-coated vesicle NP_037041 Aqp2 component cytoplasm NP_037041 Aqp2 component early endosome NP_037041 Aqp2 component exocytic vesicle NP_037041 Aqp2 component integral to membrane NP_037041 Aqp2 component lysosome NP_037041 Aqp2 component membrane fraction NP_037041 Aqp2 component membrane fraction NP_037041 Aqp2 component plasma membrane NP_037041 Aqp2 component plasma membrane NP_037041 Aqp2 component protein complex NP_037041 Aqp2 component recycling endosome NP_037041 Aqp2 component recycling endosome NP_037041 Aqp2 component rough endoplasmic reticulum NP_037041 Aqp2 component trans-Golgi network NP_037041 Aqp2 function actin binding NP_037041 Aqp2 function protein binding NP_037041 Aqp2 function transporter activity NP_037041 Aqp2 function water channel activity NP_037041 Aqp2 function water channel activity NP_037041 Aqp2 function water channel activity NP_037041 Aqp2 process actin filament depolymerization NP_037041 Aqp2 process aging NP_037041 Aqp2 process apoptosis NP_037041 Aqp2 process cell volume homeostasis NP_037041 Aqp2 process cellular response to water deprivation NP_037041 Aqp2 process cellular response to water deprivation NP_037041 Aqp2 process female pregnancy NP_037041 Aqp2 process hyperosmotic response NP_037041 Aqp2 process positive regulation of calcium ion transport NP_037041 Aqp2 process renal water transport NP_037041 Aqp2 process renal water transport NP_037041 Aqp2 process response to calcium ion NP_037041 Aqp2 process response to glucagon stimulus NP_037041 Aqp2 process response to hormone stimulus NP_037041 Aqp2 process response to lipopolysaccharide NP_037041 Aqp2 process response to lithium ion NP_037041 Aqp2 process response to salt stress NP_037041 Aqp2 process response to starvation NP_037041 Aqp2 process response to water deprivation NP_037041 Aqp2 process transport NP_037041 Aqp2 process water homeostasis NP_037041 Aqp2 process water transport NP_037041 Aqp2 process water transport NP_037041 Aqp2 process water transport NP_062220 Slc14a2 component integral to membrane NP_062220 Slc14a2 function copper ion binding NP_062220 Slc14a2 function protein binding NP_062220 Slc14a2 function urea transmembrane transporter activity NP_062220 Slc14a2 function urea transmembrane transporter activity NP_062220 Slc14a2 function urea transmembrane transporter activity NP_062220 Slc14a2 process oxidation reduction NP_062220 Slc14a2 process response to water deprivation NP_062220 Slc14a2 process urea transport NP_062220 Slc14a2 process urea transport NP_445778 Pdlim5 component Z disc NP_445778 Pdlim5 component actin cytoskeleton NP_445778 Pdlim5 component cytosol NP_445778 Pdlim5 component membrane fraction NP_445778 Pdlim5 function actin binding NP_445778 Pdlim5 function actinin binding NP_445778 Pdlim5 function metal ion binding NP_445778 Pdlim5 function protein N-terminus binding NP_445778 Pdlim5 function protein binding NP_445778 Pdlim5 function protein kinase C binding NP_445778 Pdlim5 function receptor signaling complex scaffold activity NP_445778 Pdlim5 function zinc ion binding NP_445778 Pdlim5 process heart development NP_445809 Ctnnb1 component Axin-APC-beta-catenin-GSK3B complex NP_445809 Ctnnb1 component Z disc NP_445809 Ctnnb1 component Z disc NP_445809 Ctnnb1 component adherens junction NP_445809 Ctnnb1 component apical junction complex NP_445809 Ctnnb1 component apical junction complex NP_445809 Ctnnb1 component apical part of cell NP_445809 Ctnnb1 component apical part of cell NP_445809 Ctnnb1 component basolateral plasma membrane NP_445809 Ctnnb1 component basolateral plasma membrane NP_445809 Ctnnb1 component beta-catenin destruction complex NP_445809 Ctnnb1 component beta-catenin-TCF7L2 complex NP_445809 Ctnnb1 component catenin complex NP_445809 Ctnnb1 component cell projection membrane NP_445809 Ctnnb1 component cell-cell adherens junction NP_445809 Ctnnb1 component cell-cell adherens junction NP_445809 Ctnnb1 component cell-cell junction NP_445809 Ctnnb1 component centrosome NP_445809 Ctnnb1 component cytoplasm NP_445809 Ctnnb1 component cytoplasm NP_445809 Ctnnb1 component cytoplasm NP_445809 Ctnnb1 component cytoskeleton NP_445809 Ctnnb1 component cytosol NP_445809 Ctnnb1 component cytosol NP_445809 Ctnnb1 component dendritic shaft NP_445809 Ctnnb1 component fascia adherens NP_445809 Ctnnb1 component fascia adherens NP_445809 Ctnnb1 component lamellipodium NP_445809 Ctnnb1 component lamellipodium NP_445809 Ctnnb1 component lateral plasma membrane NP_445809 Ctnnb1 component lateral plasma membrane NP_445809 Ctnnb1 component membrane NP_445809 Ctnnb1 component membrane fraction NP_445809 Ctnnb1 component membrane fraction NP_445809 Ctnnb1 component microvillus membrane NP_445809 Ctnnb1 component microvillus membrane NP_445809 Ctnnb1 component neuron projection NP_445809 Ctnnb1 component nucleus NP_445809 Ctnnb1 component nucleus NP_445809 Ctnnb1 component nucleus NP_445809 Ctnnb1 component plasma membrane NP_445809 Ctnnb1 component plasma membrane NP_445809 Ctnnb1 component protein complex NP_445809 Ctnnb1 component synapse NP_445809 Ctnnb1 component transcription factor complex NP_445809 Ctnnb1 component transcription factor complex NP_445809 Ctnnb1 function DNA binding NP_445809 Ctnnb1 function I-SMAD binding NP_445809 Ctnnb1 function alpha-catenin binding NP_445809 Ctnnb1 function alpha-catenin binding NP_445809 Ctnnb1 function cadherin binding NP_445809 Ctnnb1 function cadherin binding NP_445809 Ctnnb1 function cadherin binding NP_445809 Ctnnb1 function cadherin binding NP_445809 Ctnnb1 function chromatin binding NP_445809 Ctnnb1 function chromatin binding NP_445809 Ctnnb1 function double-stranded DNA binding NP_445809 Ctnnb1 function double-stranded DNA binding NP_445809 Ctnnb1 function kinase binding NP_445809 Ctnnb1 function promoter binding NP_445809 Ctnnb1 function protein C-terminus binding NP_445809 Ctnnb1 function protein binding NP_445809 Ctnnb1 function protein binding NP_445809 Ctnnb1 function protein binding NP_445809 Ctnnb1 function protein phosphatase binding NP_445809 Ctnnb1 function protein phosphatase binding NP_445809 Ctnnb1 function transcription activator activity NP_445809 Ctnnb1 function transcription coactivator activity NP_445809 Ctnnb1 function transcription coactivator activity NP_445809 Ctnnb1 function transcription factor activity NP_445809 Ctnnb1 function transcription factor activity NP_445809 Ctnnb1 function transcription factor binding NP_445809 Ctnnb1 function transcription factor binding NP_445809 Ctnnb1 process Schwann cell proliferation NP_445809 Ctnnb1 process T cell differentiation in the thymus NP_445809 Ctnnb1 process T cell differentiation in the thymus NP_445809 Ctnnb1 process Wnt receptor signaling pathway NP_445809 Ctnnb1 process Wnt receptor signaling pathway through beta-catenin NP_445809 Ctnnb1 process Wnt receptor signaling pathway through beta-catenin NP_445809 Ctnnb1 process anterior/posterior axis specification NP_445809 Ctnnb1 process anterior/posterior axis specification NP_445809 Ctnnb1 process apoptosis NP_445809 Ctnnb1 process apoptosis NP_445809 Ctnnb1 process bone resorption NP_445809 Ctnnb1 process bone resorption NP_445809 Ctnnb1 process camera-type eye morphogenesis NP_445809 Ctnnb1 process camera-type eye morphogenesis NP_445809 Ctnnb1 process cell differentiation NP_445809 Ctnnb1 process cell fate determination NP_445809 Ctnnb1 process cell fate specification NP_445809 Ctnnb1 process cell fate specification NP_445809 Ctnnb1 process cell maturation NP_445809 Ctnnb1 process cell maturation NP_445809 Ctnnb1 process cell morphogenesis involved in differentiation NP_445809 Ctnnb1 process cell morphogenesis involved in differentiation NP_445809 Ctnnb1 process cell proliferation NP_445809 Ctnnb1 process cell-cell adhesion NP_445809 Ctnnb1 process cell-cell adhesion NP_445809 Ctnnb1 process cell-cell adhesion NP_445809 Ctnnb1 process cell-matrix adhesion NP_445809 Ctnnb1 process cell-matrix adhesion NP_445809 Ctnnb1 process cellular process NP_445809 Ctnnb1 process cellular protein localization NP_445809 Ctnnb1 process cellular protein localization NP_445809 Ctnnb1 process dorsal/ventral axis specification NP_445809 Ctnnb1 process dorsal/ventral axis specification NP_445809 Ctnnb1 process dorsal/ventral pattern formation NP_445809 Ctnnb1 process ectoderm development NP_445809 Ctnnb1 process ectoderm development NP_445809 Ctnnb1 process embryonic arm morphogenesis NP_445809 Ctnnb1 process embryonic arm morphogenesis NP_445809 Ctnnb1 process embryonic digit morphogenesis NP_445809 Ctnnb1 process embryonic digit morphogenesis NP_445809 Ctnnb1 process embryonic hindlimb morphogenesis NP_445809 Ctnnb1 process embryonic hindlimb morphogenesis NP_445809 Ctnnb1 process endoderm formation NP_445809 Ctnnb1 process endodermal cell fate commitment NP_445809 Ctnnb1 process endodermal cell fate commitment NP_445809 Ctnnb1 process epithelial cell differentiation involved in prostate gland development NP_445809 Ctnnb1 process forebrain development NP_445809 Ctnnb1 process forebrain development NP_445809 Ctnnb1 process gastrulation with mouth forming second NP_445809 Ctnnb1 process gastrulation with mouth forming second NP_445809 Ctnnb1 process glial cell fate determination NP_445809 Ctnnb1 process glial cell fate determination NP_445809 Ctnnb1 process heart development NP_445809 Ctnnb1 process heart development NP_445809 Ctnnb1 process hemopoiesis NP_445809 Ctnnb1 process hemopoiesis NP_445809 Ctnnb1 process liver development NP_445809 Ctnnb1 process lung development NP_445809 Ctnnb1 process lung development NP_445809 Ctnnb1 process midgut development NP_445809 Ctnnb1 process morphogenesis of embryonic epithelium NP_445809 Ctnnb1 process morphogenesis of embryonic epithelium NP_445809 Ctnnb1 process myoblast differentiation NP_445809 Ctnnb1 process negative regulation of cell differentiation NP_445809 Ctnnb1 process negative regulation of chondrocyte differentiation NP_445809 Ctnnb1 process negative regulation of chondrocyte differentiation NP_445809 Ctnnb1 process negative regulation of osteoclast differentiation NP_445809 Ctnnb1 process negative regulation of osteoclast differentiation NP_445809 Ctnnb1 process negative regulation of transcription from RNA polymerase II promoter NP_445809 Ctnnb1 process negative regulation of transcription from RNA polymerase II promoter NP_445809 Ctnnb1 process negative regulation of transcription, DNA-dependent NP_445809 Ctnnb1 process odontogenesis of dentine-containing tooth NP_445809 Ctnnb1 process odontogenesis of dentine-containing tooth NP_445809 Ctnnb1 process odontogenesis of dentine-containing tooth NP_445809 Ctnnb1 process osteoclast differentiation NP_445809 Ctnnb1 process pancreas development NP_445809 Ctnnb1 process pancreas development NP_445809 Ctnnb1 process patterning of blood vessels NP_445809 Ctnnb1 process patterning of blood vessels NP_445809 Ctnnb1 process positive regulation of MAPKKK cascade NP_445809 Ctnnb1 process positive regulation of MAPKKK cascade NP_445809 Ctnnb1 process positive regulation of epithelial cell differentiation NP_445809 Ctnnb1 process positive regulation of epithelial cell differentiation NP_445809 Ctnnb1 process positive regulation of epithelial cell proliferation involved in prostate gland development NP_445809 Ctnnb1 process positive regulation of gene-specific transcription from RNA polymerase II promoter NP_445809 Ctnnb1 process positive regulation of heparan sulfate proteoglycan biosynthetic process NP_445809 Ctnnb1 process positive regulation of osteoblast differentiation NP_445809 Ctnnb1 process positive regulation of osteoblast differentiation NP_445809 Ctnnb1 process positive regulation of transcription NP_445809 Ctnnb1 process positive regulation of transcription from RNA polymerase II promoter NP_445809 Ctnnb1 process positive regulation of transcription from RNA polymerase II promoter NP_445809 Ctnnb1 process positive regulation of transcription, DNA-dependent NP_445809 Ctnnb1 process protein heterooligomerization NP_445809 Ctnnb1 process proximal/distal pattern formation NP_445809 Ctnnb1 process proximal/distal pattern formation NP_445809 Ctnnb1 process regulation of cell differentiation NP_445809 Ctnnb1 process regulation of cell proliferation NP_445809 Ctnnb1 process regulation of cell proliferation NP_445809 Ctnnb1 process regulation of centriole-centriole cohesion NP_445809 Ctnnb1 process regulation of epithelial cell differentiation NP_445809 Ctnnb1 process regulation of osteoblast differentiation NP_445809 Ctnnb1 process regulation of osteoclast differentiation NP_445809 Ctnnb1 process regulation of transcription NP_445809 Ctnnb1 process regulation of transcription, DNA-dependent NP_445809 Ctnnb1 process response to cadmium ion NP_445809 Ctnnb1 process response to cytokine stimulus NP_445809 Ctnnb1 process response to estrogen stimulus NP_445809 Ctnnb1 process response to hormone stimulus NP_445809 Ctnnb1 process response to organic cyclic substance NP_445809 Ctnnb1 process skeletal system development NP_445809 Ctnnb1 process skeletal system development NP_445809 Ctnnb1 process synapse organization NP_445809 Ctnnb1 process synapse organization NP_445809 Ctnnb1 process synaptic transmission NP_445809 Ctnnb1 process synaptic transmission NP_445809 Ctnnb1 process synaptic vesicle transport NP_445809 Ctnnb1 process synaptic vesicle transport NP_445809 Ctnnb1 process thymus development NP_445809 Ctnnb1 process thymus development NP_690003 Carhsp1 component cytoplasm NP_690003 Carhsp1 component cytosol NP_690003 Carhsp1 function DNA binding NP_690003 Carhsp1 function protein binding NP_690003 Carhsp1 function protein binding NP_690003 Carhsp1 function protein binding NP_690003 Carhsp1 process calcium-mediated signaling NP_690003 Carhsp1 process regulation of transcription, DNA-dependent NP_998791 kif13B component microtubule NP_998791 kif13B function ATP binding NP_998791 kif13B function microtubule motor activity NP_998791 kif13B function nucleotide binding NP_998791 kif13B process microtubule-based movement XP_001078032 Ahnak component not classified XP_001078032 Ahnak function protein binding XP_001078032 Ahnak process not classified XP_215763 Tnks1bp1 component not classified XP_215763 Tnks1bp1 function not classified XP_215763 Tnks1bp1 process not classified XP_225259 Dsp component adherens junction XP_225259 Dsp component basolateral plasma membrane XP_225259 Dsp component basolateral plasma membrane XP_225259 Dsp component cornified envelope XP_225259 Dsp component desmosome XP_225259 Dsp component desmosome XP_225259 Dsp component desmosome XP_225259 Dsp component fascia adherens XP_225259 Dsp component mitochondrion XP_225259 Dsp component mitochondrion XP_225259 Dsp component plasma membrane XP_225259 Dsp function protein binding, bridging XP_225259 Dsp process keratinocyte differentiation XP_225259 Dsp process peptide cross-linking XP_225259 Dsp process wound healing

Statistic: component basolateral plasma 5 cytoplasm 5 membrane fraction 5 plasma membrane 5 not classified 4 cytosol 4 apical plasma mem 3 nucleus 3 desmosome 3 Z disc 3 fascia adherens 3 transcription factor 2 lamellipodium 2 membrane 2 mitochondrion 2 microvillus membra 2 integral to membran 2 cell-cell adherens ju 2 protein complex 2 recycling endosome 2 lateral plasma mem 2 adherens junction 2 apical part of cell 2 apical junction com 2 dendritic shaft 1 Axin-APC-beta-cate 1 synapse 1 actin cytoskeleton 1 beta-catenin destru 1 trans-Golgi network 1 rough endoplasmic 1 beta-catenin-TCF7L 1 catenin complex 1 microtubule 1 cornified envelope 1 lysosome 1 cell projection mem 1 exocytic vesicle 1 early endosome 1 cell-cell junction 1 clathrin-coated vesi 1 cell fraction 1 centrosome 1 neuron projection 1 cytoskeleton 1 Total = 90

Statistic: function protein binding 11 cadherin binding 4 not classified 3 water channel activ 3 urea transmembran 3 transcription factor 2 ATP binding 2 protein phosphatas 2 nucleotide binding 2 actin binding 2 transcription factor 2 double-stranded DN 2 chromatin binding 2 transcription coactiv 2 alpha-catenin bindin 2 DNA binding 2 transcription activat 1 zinc ion binding 1 receptor signaling c 1 protein kinase C bin 1 protein N-terminus 1 metal ion binding 1 actinin binding 1 I-SMAD binding 1 copper ion binding 1 microtubule motor a 1 transporter activity 1 kinase binding 1 transferase activity 1 protein binding, brid 1 promoter binding 1 cyclin-dependent pr 1 protein C-terminus 1 Total = 63

Statistic: process not classified 3 apoptosis 3 water transport 3 heart development 3 cell-cell adhesion 3 odontogenesis of d 3 Wnt receptor signal 2 synaptic vesicle tra 2 synaptic transmissi 2 synapse organizatio 2 skeletal system dev 2 cellular response to 2 renal water transpo 2 response to hormon 2 response to water d 2 regulation of transc 2 urea transport 2 T cell differentiation 2 Wnt receptor signal 2 anterior/posterior ax 2 bone resorption 2 regulation of cell pr 2 camera-type eye m 2 proximal/distal patte 2 cell fate specificatio 2 cell maturation 2 positive regulation o 2 cell morphogenesis 2 positive regulation o 2 cell-matrix adhesion 2 cellular protein loca 2 dorsal/ventral axis s 2 positive regulation o 2 positive regulation o 2 patterning of blood 2 pancreas developm 2 ectoderm developm 2 thymus developmen 2 embryonic arm mor 2 negative regulation 2 negative regulation 2 negative regulation 2 embryonic digit mor 2 embryonic hindlimb 2 morphogenesis of e 2 endodermal cell fat 2 lung development 2 forebrain developm 2 hemopoiesis 2 glial cell fate determ 2 gastrulation with mo 2 response to organic 1 epithelial cell differe 1 liver development 1 endoderm formation 1 midgut developmen 1 myoblast differentia 1 negative regulation 1 negative regulation 1 dorsal/ventral patte 1 osteoclast differenti 1 positive regulation o 1 cellular process 1 positive regulation o 1 calcium-mediated s 1 cell proliferation 1 positive regulation o 1 positive regulation o 1 positive regulation o 1 cell fate determinat 1 cell differentiation 1 protein heterooligom 1 regulation of cell dif 1 regulation of centrio 1 regulation of epithe 1 regulation of osteob 1 Schwann cell prolife 1 microtubule-based 1 regulation of osteoc 1 oxidation reduction 1 keratinocyte differe 1 water homeostasis 1 transport 1 regulation of transc 1 response to starvat 1 response to salt str 1 response to lithium 1 response to lipopoly 1 response to cadmiu 1 response to glucag 1 response to calcium 1 response to cytokin 1 positive regulation o 1 hyperosmotic respo 1 female pregnancy 1 response to estroge 1 cell volume homeos 1 peptide cross-linkin 1 aging 1 actin filament depol 1 wound healing 1 protein amino acid 1 Total = 159 Input Protein Name CDD Conserved Domain NP_001019962 epsin 3 cd03571 ENTH_epsin NP_001019962 epsin 3 PRK07764 PRK07764 NP_001101454 PDLIM1 interacti cd00180 S_TKc NP_001101454 PDLIM1 interacti smart00220 S_TKc NP_001101454 PDLIM1 interacti cd00180 S_TKc NP_001101454 PDLIM1 interacti smart00220 S_TKc NP_001101454 PDLIM1 interacti cd00180 S_TKc NP_001101454 PDLIM1 interacti smart00220 S_TKc NP_001101454 PDLIM1 interacti cd00180 S_TKc NP_001101454 PDLIM1 interacti smart00220 S_TKc NP_001124461 prostaglandin-E scd00237 p23 NP_001129068 ArfGAP with FG cl02464 ArfGap NP_037041 aquaporin 2 (coll cd00333 MIP NP_110484 Yamaguchi sarcocd00173 SH2 NP_110484 Yamaguchi sarcocd00174 SH3 NP_110484 Yamaguchi sarcopfam07714 Pkinase_Tyr NP_110484 Yamaguchi sarcocd05034 PTKc_Src_like NP_445747 calpastatin isoforpfam00748 Calpain_inhib NP_446225 tight junction protcd00992 PDZ_signaling NP_446225 tight junction protsmart00072 GuKc NP_446225 tight junction protcl09099 P-loop NTPase NP_446225 tight junction protcl09950 SH3 NP_742005 calnexin precursopfam00262 Calreticulin NP_786937 heat shock prote pfam00183 HSP90 NP_786937 heat shock prote cl00075 HATPase_c NP_786941 PC4 and SFRS1 cd05834 HDGF_related NP_789826 septin 9 isoform cd01850 CDC_Septin NP_808877 solute carrier famcl01829 UT NP_835196 ATP-binding cas cd03263 ABC_subfamily_A NP_835196 ATP-binding cas TIGR01257 rim_protein XP_001053711 pfam09162 Tap-RNA_bind XP_001053711 cl04338 TAP_C XP_001053711 cl09109 NTF2_like XP_001059640 cd00183 TFIIS_I XP_001059640 TIGR01385 TFSII XP_001059640 cl02609 TFIIS_C XP_001059640 cl02637 TFIIS_M XP_001059640 cd00183 TFIIS_I XP_001059640 TIGR01385 TFSII XP_001059640 cl02609 TFIIS_C XP_001059640 cl02637 TFIIS_M XP_001071043 cd00992 PDZ_signaling XP_001071043 cd01258 PH_syntrophin XP_001071043 cd00992 PDZ_signaling XP_001071043 cd01258 PH_syntrophin XP_237286 cd00173 SH2 XP_237286 pfam10409 PTEN_C2 XP_237286 cl00053 PTPc XP_237286 cl00273 PH-like XP_237286 cd00173 SH2 XP_237286 pfam10409 PTEN_C2 XP_237286 cl00053 PTPc XP_237286 cl00273 PH-like XP_341257 pfam10409 PTEN_C2 XP_341257 cl00053 PTPc XP_341257 cd00173 SH2 XP_341257 cl00273 PH-like XP_341257 cd00029 C1 XP_341257 cd00173 SH2 XP_341257 cd01213 tensin XP_341257 pfam10409 PTEN_C2 XP_341257 cl00053 PTPc

Statistic: conserved domain S_TKc 8 SH2 5 PTEN_C2 4 PTPc 4 PDZ_signaling 3 PH-like 3 SH3 2 TFIIS_M 2 TFIIS_C 2 TFSII 2 TFIIS_I 2 PH_syntrophin 2 ENTH_epsin 1 Tap-RNA_bind 1 rim_protein 1 ABC_subfamily_A 1 UT 1 CDC_Septin 1 HDGF_related 1 HATPase_c 1 HSP90 1 Calreticulin 1 P-loop NTPase 1 GuKc 1 TAP_C 1 Calpain_inhib 1 PTKc_Src_like 1 Pkinase_Tyr 1 NTF2_like 1 C1 1 MIP 1 ArfGap 1 p23 1 tensin 1 PRK07764 1 Total = 62 Input Protein Name CDD Conserved Domain NP_001019932 leucine rich repeapfam09738 DUF2051 NP_001093976 PCTAIRE protein cd00180 S_TKc NP_001093976 PCTAIRE protein smart00220 S_TKc NP_001093976 PCTAIRE protein cd00180 S_TKc NP_001093976 PCTAIRE protein smart00220 S_TKc NP_001093976 PCTAIRE protein cd00180 S_TKc NP_001093976 PCTAIRE protein smart00220 S_TKc NP_001101892 plexin domain concl02601 PSI NP_001101892 plexin domain concl02648 NIDO NP_001101892 plexin domain concl02601 PSI NP_001101892 plexin domain concl02648 NIDO NP_001101892 plexin domain concl02601 PSI NP_001101892 plexin domain concl02648 NIDO NP_037041 aquaporin 2 (collecd00333 MIP NP_062220 solute carrier fam cl01829 UT NP_062220 solute carrier fam cl01829 UT NP_445778 PDZ and LIM domcd00992 PDZ_signaling NP_445778 PDZ and LIM domcl02475 LIM NP_445809 beta-catenin cd00020 ARM NP_445809 beta-catenin cl02500 ARM NP_690003 calcium regulated cd04458 CSP_CDS NP_998791 kinesin 13B cd00060 FHA NP_998791 kinesin 13B cd01365 KISc_KIF1A_KIF1B NP_998791 kinesin 13B pfam01302 CAP_GLY XP_215763 null null XP_225259 COG1196 Smc XP_225259 cl02531 Plectin XP_225259 cd00176 SPEC XP_225259 TIGR02168 SMC_prok_B XP_225259 cl02531 Plectin XP_225259 COG1196 Smc XP_225259 cl02531 Plectin XP_225259 cd00176 SPEC XP_225259 TIGR02168 SMC_prok_B XP_225259 cl02531 Plectin

Statistic: conserved domain S_TKc 6 Plectin 4 PSI 3 NIDO 3 SMC_prok_B 2 SPEC 2 Smc 2 UT 2 ARM 2 DUF2051 1 CSP_CDS 1 LIM 1 PDZ_signaling 1 FHA 1 MIP 1 KISc_KIF1A_KIF 1 CAP_GLY 1 null 1 Total = 35 Supplementary Table 1: List of phosphopeptides quantified by mass spectrometry that did not change significantly with dDAVP exposure. Mean and standard error (SE) values are calculated from log2(D/C), where D and C are areas of the reconstructed ion chromatograms for dDAVP- treated and control samples, respectively. Note: Phosphorylation sites were not scored, despite confidence in phosphopeptide identification. # Name Sequence Accession # Mean ± SE 1 NFkB interacting protein 1 R.IPMPPSSPQPRS*T*PRQRPIPLS*MIFK.L(+3) XP_001075705 -0.56 ± 0.24 2 hsp 90kDa alpha (cytosolic), class B member 1 K.IEDVGS*DEEDDSGKDKK.K(+2) NP_001004082 -0.55 ± 0.35 3 EPS8-like 2 R.QSILPPPQS*PAPIPFQRQPGDS*PQAK.N(+3) NP_001101978 -0.55 ± 0.34 4 scaffold attachment factor B R.APTAAPS*PEPR.D(+2) NP_071789 -0.53 ± 0.23 5 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.MEGPGVDIDS*PDVNIEGPEGK.L(+3) XP_001078032 -0.49 ± 0.17 6 calnexin K.AEEDEILNRS*PR.N(+2) NP_742005 -0.48 ± 0.25 7 PREDICTED: PDZ domain containing 10 K.QSATT*LLVGPRHGISHVIDLK.T(+3) XP_001053582 -0.48 ± 0.7 8 PREDICTED: PDZ-domain protein scribble R.MVEPENAVTITPLRPEDDY*SPR.E(+3) XP_001074724 -0.48 ± 0.41 9 PREDICTED: serine/arginine repetitive matrix 2 K.AAEIPAVASCWVGPQVS*PEHK.E(+3) XP_001056343 -0.47 ± 0.17 10 thyroid hormone receptor associated protein 3 R.ASVSDLS*PR.E(+2) NP_001009693 -0.45 ± 0.08 oxidative-stress responsive 1 R.LHKTEDGGWEWS*DDEFDEESEEGKAAISQLR.S(+ NP_001101664 -0.45 ± 1.59 11 4) 12 PREDICTED: tensin R.HLGGSGSVVPGS*PSLDR.H(+2) XP_237286 -0.44 ± 0.05 13 lethal giant larvae homolog 2 R.S*QSDGAETKPGPVMEHALLSDAWVLK.E(+3) NP_001121021 -0.43 ± 0.64 monocarboxylate transporter R.SIIQIYLTT*GVIT*GLGLALNFQPS*LIMLNRYFNK.R( NP_110461 -0.43 ± 0.73 14 +4) PREDICTED: Epithelial protein lost in neoplasm (mEPLIN) R.TSSVKS*PKPLS*PSLRK.G(+3) XP_217039 -0.43 ± 0.1 15 LIM domain containing preferred translocation partner in R.RSS*LDAEIDSLTSILADLECSSPYKPR.A(+3) NP_001013886 -0.42 ± 1.05 16 lipoma 17 Ste-20 related kinase K.TEDGDWEWS*DDEMDEKSEEGK.A(+3) NP_062235 -0.42 ± 0.65 PREDICTED: GrpE protein homolog 2, mitochondrial R.S*LWAVQRLQRLLASGAVS*ES*R.G(+3) XP_001058643 -0.41 ± 0.92 18 precursor (Mt-GrpE#2) 19 matrin 3 R.RDS*FDDRGPSLNPVLDYDHGSR.S(+3) NP_062022 -0.4 ± 0.3 PREDICTED: CAP-binding protein complex interacting protein K.S*RPVALGRVQK.V(+2) XP_001077962 -0.4 ± 0.39 20 1 21 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.MYFPDVEFDIKS*PK.F(+3) XP_001078032 -0.4 ± 0.13 22 actin-binding LIM protein 1 R.TLS*PTPSAEGFQDGR.D(+2) NP_001037859 -0.39 ± 0.21 ladinin R.LPS*VEEAEVSKPSPPASKDEGEEFQAILR.T(+3) NP_001101412 -0.39 ± 0.62 23

24 tight junction protein 3 R.AIAEPES*PGESR.Y(+2) NP_001101543 -0.39 ± 0.15 25 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.FKAEAALPS*PK.L(+2) XP_001078032 -0.39 ± 0.12 26 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.MEGPGVDIDS*PDVNIEGPEGK.L(+2) XP_001078032 -0.39 ± 0.09 27 PREDICTED: PDZ-domain protein scribble R.MVEPENAVTITPLRPEDDYS*PR.E(+3) XP_001074724 -0.38 ± 0.53 28 periplakin R.S*IVVIDPDTGRELSPEEAHR.A(+3) NP_001100446 -0.37 ± 0.21 protein C K.TIVPICLPNSGLAQELSQAGQETVVT*GWGY*QS*D NP_036935 -0.37 ± 0.45 29 K.V(+4) 30 endosulfine alpha isoform 2 K.YFDS*GDYNMAK.A(+2) NP_068614 -0.37 ± 0.12 septin 2 R.ILDEIEEHSIKIYHLPDAES*DEDEDFKEQTR.L(+4) NP_476489 -0.37 ± 0.74 31

32 PREDICTED: neuron navigator 1 K.GQLTNIVSPT*AAT*TPR.I(+3) XP_001063421 -0.37 ± 0.17 33 PREDICTED: tensin R.AINPTMAAPGS*PSLSHR.Q(+3) XP_237286 -0.37 ± 0.17 34 calnexin K.AEEDEILNRS*PR.N(+3) NP_742005 -0.36 ± 0.17 35 PREDICTED: tensin R.TVGTNTPPS*PGFGR.R(+2) XP_237286 -0.36 ± 0.13 heat shock protein 90kDa alpha (cytosolic), class B member 1 K.IEDVGS*DEEDDSGKDK.K(+2) NP_001004082 -0.35 ± 0.31 36

37 mitogen-activated protein kinase 6 R.ES*ADVVDKLNDLNSSVSQLEM#KSLISK.S(+3) NP_113810 -0.35 ± 0.15 38 EPS8-like 2 K.HSLSSEPQVPEDVAPPGS*SPHANR.G(+3) NP_001101978 -0.34 ± 0.04 39 EPS8-like 2 K.HSLSSEPQVPEDVAPPGSS*PHANR.G(+3) NP_001101978 -0.34 ± 0.04 PREDICTED: Epithelial protein lost in neoplasm (mEPLIN) K.GENEETLGRPAQPPSAGETPHS*PGVEDAPIAK.V( XP_217039 -0.34 ± 0.23 40 +3) PREDICTED: Epithelial protein lost in neoplasm (mEPLIN) K.GWSEPEPEQSEEFGGGTVTQTES*PRPSR.E(+3) XP_217039 -0.34 ± 0.38 41 PREDICTED: tensin-like SH2 domain containing 1 R.WDS*YENMSADGEVLHTQGPVDGSLYAK.V(+3) XP_341257 -0.34 ± 0.5 42

43 BCL2-associated transcription factor 1 R.IDIS*PSALR.K(+2) NP_001041317 -0.33 ± 0.18 44 mannose receptor, C type 1 K.GSSKAAGVVIVVLLIVIGAGVAAY*FFY*KK.R(+3) NP_001099593 -0.33 ± 0.36 45 hypothetical protein LOC300783 R.WLDES*DAEMELR.A(+2) NP_001100301 -0.33 ± 0.26 46 fibronectin leucine rich transmembrane protein 1 R.NS*LAAPPLNLPSAHLQK.L(+2) NP_001102630 -0.32 ± 0.78 47 heterogeneous nuclear ribonucleoprotein K R.RDYDDMS*PR.R(+2) NP_476482 -0.32 ± 0.05 48 PCTAIRE protein kinase 3 R.RAS*LSDIGFGK.L(+2) NP_001093976 -0.31 ± 0.49 49 actinin, alpha 1 K.NY*ITGDELR.R(+2) NP_112267 -0.31 ± 0.34 50 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.VPDVDIS*SPGVNVEAPDIHVK.A(+3) XP_001078032 -0.31 ± 0.27 51 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.VPDVDISS*PGVNVEAPDIHVK.A(+3) XP_001078032 -0.31 ± 0.27 PREDICTED: Ras-GTPase-activating protein binding protein K.SAS*PAPADVAPAQEDLR.T(+2) XP_340803 -0.31 ± 0.07 52 1 heat shock protein 90kDa alpha (cytosolic), class B member 1 K.IEDVGS*DEEDDSGKDK.K(+3) NP_001004082 -0.3 ± 0.3 53

54 microtubule-associated protein 4 K.VGS*LDNVGHLPAGGTVK.T(+3) NP_001019449 -0.3 ± 0.07 55 PREDICTED: tensin R.HLGGSGSVVPGS*PSLDR.H(+3) XP_237286 -0.3 ± 0.08 56 ADNP homeobox 2 K.ERHHVMPT*VHT*MLR.S(+2) NP_001120845 -0.29 ± 0.17 57 calcium regulated heat stable protein 1 R.DRS*PS*PLRGNVVPS*PLPTR.R(+3) NP_690003 -0.29 ± 0.2 58 calcium regulated heat stable protein 1 R.DRS*PS*PLRGNVVPSPLPT*R.R(+3) NP_690003 -0.29 ± 0.2 59 PREDICTED: cyclin fold protein 1 R.S*ASADNLILPR.W(+2) XP_341546 -0.29 ± 0.1 aquaporin 4 isoform 1 K.GSYMEVEDNRS*QVETEDLILKPGVVHVIDIDR.G(+ NP_036957 -0.28 ± 0.4 60 4) 61 PREDICTED: ribosomal protein L27a K.LWTLVS*KQTLVSAAKNK.T(+3) XP_001055169 -0.28 ± 0.2 62 NEL-like 1 R.CVSDPCLAGNIAY*DIR.K(+2) NP_112331 -0.27 ± 0.15 63 PREDICTED: serine/arginine repetitive matrix 2 K.ELS*HSPPRDNSFESLEFR.N(+3) XP_001056343 -0.27 ± 0.24 64 PREDICTED: serine/arginine repetitive matrix 2 K.ELSHS*PPRDNSFESLEFR.N(+3) XP_001056343 -0.27 ± 0.24 PREDICTED: Epithelial protein lost in neoplasm (mEPLIN) K.GENEETLGRPAQPPSAGETPHS*PGVEDAPIAK.V( XP_217039 -0.27 ± 0.16 65 +4) eukaryotic translation initiation factor 4B K.SLENETLNKEEDCHS*PTSKPPKPDQPLK.V(+4) NP_001008325 -0.26 ± 0.15 66 eukaryotic translation initiation factor 4B K.SLENETLNKEEDCHSPT*SKPPKPDQPLK.V(+4) NP_001008325 -0.26 ± 0.15 67

68 RNA binding motif protein 39 K.DKS*PVREPIDNLTPEER.D(+3) NP_001013225 -0.26 ± 0.2 solute carrier family 9 (sodium/hydrogen exchanger), member R.IGS*DPLAYEPK.A(+2) NP_036784 -0.26 ± 0.19 69 1 70 phosphatidylethanolamine binding protein K.FKVES*FR.K(+2) NP_058932 -0.26 ± 0.34 71 synaptosomal-associated protein 23 K.ATWGDGGDSS*PSNVVSK.Q(+2) NP_073180 -0.26 ± 0.17 72 PREDICTED: putative pheromone receptor (Go-VN3) K.HQRRY*LIVLT*GPPWLTS*YK.L(+3) XP_001060597 -0.26 ± 0.39 73 PREDICTED: tankyrase 1-binding protein of 182 kDa K.ENYEDQEPLAGQES*PITLATR.E(+3) XP_215763 -0.26 ± 0.07 74 PREDICTED: cyclin fold protein 1 R.SAS*ADNLILPR.W(+2) XP_341546 -0.26 ± 0.12 myeloid/lymphoid (trithorax homolog, Drosophila); K.QGAIYHGLATLLNQPS*PMMQR.I(+3) NP_037349 -0.25 ± 0.21 75 translocated to, 4 76 heat shock protein 1 R.QLS*SGVSEIR.Q(+2) NP_114176 -0.25 ± 0.09 PREDICTED: Syntaxin-binding protein 5-like (Tomosyn-2) R.GQRLGELEEKTAGMMT*SAEAFSK.H(+2) XP_001070379 -0.25 ± 0.24 77

78 PREDICTED: eIF-5B isoform 2 K.SVPTIDS*GNEDDDSSFK.I(+2) XP_001071547 -0.25 ± 0.12 PREDICTED: CAP-binding protein complex interacting protein K.S*RPVALGRVQK.V(+1) XP_001077962 -0.25 ± 0.74 79 1 80 epsin 3 R.TPVLPSGPPITDPWAPSS*PTPK.L(+3) NP_001019962 -0.24 ± 0.12 81 epsin 3 R.TPVLPSGPPITDPWAPSSPT*PK.L(+3) NP_001019962 -0.24 ± 0.12 ladinin R.LPS*VEEAEVSKPSPPASKDEGEEFQAILR.T(+4) NP_001101412 -0.24 ± 0.31 82 calcium channel, voltage-dependent, alpha2/delta subunit 1 R.RRPWY*IQGAASPKDM#LILVDVS*GSVSGLTLK.L( NP_001104317 -0.24 ± 0.32 83 isoform 3 +3) 84 aquaporin 2 (collecting duct) R.RQS*VELHSPQS*LPR.G(+3) NP_037041 -0.24 ± 1.17 85 hepatoma-derived growth factor R.AGDMLEDS*PKRPK.E(+2) NP_446159 -0.24 ± 0.19 86 heterogeneous nuclear ribonucleoprotein K R.DYDDMS*PR.R(+2) NP_476482 -0.24 ± 0.04 87 alpha-spectrin 2 R.WRS*LQQLAEER.S(+2) NP_741984 -0.23 ± 0.13 PREDICTED: Oxysterol-binding protein-related protein 3 R.LHS*SNPNLSTLDFGEEK.S(+3) XP_342684 -0.23 ± 0.13 88 (ORP-3) PREDICTED: Oxysterol-binding protein-related protein 3 R.LHSS*NPNLSTLDFGEEK.S(+3) XP_342684 -0.23 ± 0.13 89 (ORP-3) 90 catenin (cadherin-associated protein), alpha 1, 102kDa R.TPEELDDS*DFETEDFDVR.S(+2) NP_001007146 -0.22 ± 0.12 solute carrier family 9 (sodium/hydrogen exchanger), member K.ITNYLTVPAHKLDS*PTMSR.A(+3) NP_036784 -0.22 ± 0.14 91 1 solute carrier family 9 (sodium/hydrogen exchanger), member K.ITNYLTVPAHKLDSPTMS*R.A(+3) NP_036784 -0.22 ± 0.14 92 1 93 myosin, heavy chain 9, non-muscle R.KGTGDCS*DEEVDGKADGADAK.A(+2) NP_037326 -0.22 ± 0.23 adenosine monophosphate deaminase 3 K.GPPSVSPAMSPT*T*PLVLGAASKPGLAPYDMPEY NP_113732 -0.22 ± 0.18 94 QR.A(+4) 95 calnexin K.QKS*DAEEDGGTGS*QDEEDSKPK.A(+3) NP_742005 -0.22 ± 0.48 ADP-ribosylation factor guanine nucleotide-exchange factor 2 R.ELEKPIQSKPQS*PVIQATAGS*PK.F(+3) NP_851597 -0.22 ± 0.08 96 (brefeldin A-inhibited) 97 PREDICTED: CG5937-PA R.GPDS*PLLQRPQHLIDQGQMR.H(+3) XP_001072524 -0.22 ± 0.19 PREDICTED: tensin K.VSSS*PVANGMAS*PSGSSTVSFSHTLPDFSK.Y(+3 XP_237286 -0.22 ± 0.25 98 ) branched chain keto acid dehydrogenase E1, alpha R.IGHHS*TSDDSSAYRS*VDEVNYWDKQDHPISR.L(+ NP_036914 -0.21 ± 0.13 99 polypeptide 4) 100 protein kinase, cAMP dependent regulatory, type II alpha R.RVS*VCAETFNPDEEEDNDPR.V(+3) NP_062137 -0.21 ± 0.07 101 drebrin-like K.LRS*PFLQK.Q(+2) NP_112642 -0.21 ± 0.06 adenosine monophosphate deaminase 3 K.GPPSVS*PAMS*PTTPLVLGAASKPGLAPYDMPEY NP_113732 -0.21 ± 0.3 102 QR.A(+3) 103 hepatoma-derived growth factor R.AGDM#LEDS*PKRPK.E(+3) NP_446159 -0.21 ± 0.13 PREDICTED: (NUANCE protein) R.LTSHTPGLDDEKEAS*ENETDIEDPREIPADSWR.K( XP_001080795 -0.21 ± 0.12 104 +4) PREDICTED: splicing factor, arginine/serine-rich 1 (ASF/SF2) R.VKVDGPRS*PSYGR.S(+3) XP_001081187 -0.21 ± 0.09 105 PREDICTED: Epithelial protein lost in neoplasm (mEPLIN) R.SEAQQPIYTKPLS*PDAR.T(+2) XP_217039 -0.21 ± 0.12 106

107 epsin 3 R.TPVLPSGPPITDPWAPS*SPTPK.L(+3) NP_001019962 -0.2 ± 0.09 108 p21-activated kinase 4 R.SNS*LRRES*PPPPAR.A(+3) NP_001099708 -0.2 ± 0.13 109 vinculin K.GWLRDPNAS*PGDAGEQAIR.Q(+3) NP_001100718 -0.2 ± 0.07 PREDICTED: NADH dehydrogenase (ubiquinone) 1 beta K.YKPAPLAALPSTLDSVEY*DVSPETRK.A(+3) XP_001056198 -0.2 ± 0.75 110 subcomplex, 4, 15kDa 111 PREDICTED: (PDHE1-A type I) isoform 1 R.Y*HGHSM#SDPGVS*YR.T(+3) XP_001060860 -0.2 ± 0.25 112 PREDICTED: (PDHE1-A type I) isoform 1 R.YHGHS*M#SDPGVS*YR.T(+3) XP_001060860 -0.2 ± 0.25 113 Cobl-like 1 R.QSS*LNFQSSDPEHIR.Q(+3) NP_001101203 -0.19 ± 0.06 114 erythrocyte protein band 4.1-like 1 isoform L R.RLPSS*PAS*PSPK.G(+2) NP_067713 -0.19 ± 0.08 115 plectin 1 R.T*QLASWSDPTEETGPVAGILDTETLEK.V(+3) NP_071796 -0.19 ± 0.28 116 plectin 1 R.TQLAS*WSDPTEETGPVAGILDTETLEK.V(+3) NP_071796 -0.19 ± 0.28 117 reticulon 4 R.GPLPAAPPAAPERQPS*WER.S(+2) NP_114019 -0.19 ± 0.17 solute carrier family 14 (urea transporter), member 2 isoform K.LYESELSS*PTWPSSSQDTHPALPLLEMPEEK.D(+3 NP_808877 -0.19 ± 0.47 118 2 ) 119 proteasome subunit alpha type 3-like K.ESLKEEDES*DDDNM#.-(+2) NP_001004094 -0.18 ± 0.13 120 catenin (cadherin-associated protein), alpha 1, 102kDa R.T*PEELDDSDFETEDFDVR.S(+3) NP_001007146 -0.18 ± 0.22 121 catenin (cadherin-associated protein), alpha 1, 102kDa R.TPEELDDS*DFETEDFDVR.S(+3) NP_001007146 -0.18 ± 0.22 122 ladinin K.GRPEETAAQQKS*PVSEKTPVSAK.R(+4) NP_001101412 -0.18 ± 0.14 123 cingulin-like 1 R.RQDS*AGPILDGAR.S(+2) NP_001101634 -0.18 ± 0.11 124 galectin-related protein K.LDDGHLNNSLGS*PVQADVYFPR.L(+3) NP_001128202 -0.18 ± 0.21 125 heterogeneous nuclear ribonucleoprotein A1 K.SES*PKEPEQLR.K(+3) NP_058944 -0.18 ± 0.24 126 PREDICTED: zinc finger CCCH-type containing 5 K.LHS*LQS*QLRLDLEAVDGVIFQLRAK.Q(+3) XP_001059160 -0.18 ± 0.25 heat shock protein 90kDa alpha (cytosolic), class B member 1 K.IEDVGS*DEEDDSGKDKK.K(+3) NP_001004082 -0.17 ± 0.21 127

128 RAS p21 protein activator (GTPase activating protein) 1 K.WPTNNTMRT*RVVS*GFVFLR.L(+3) NP_037267 -0.17 ± 0.1 129 SH3-domain kinase binding protein 1 R.S*IEVENDFLPVEK.T(+2) NP_445812 -0.17 ± 0.04 130 septin 2 K.IYHLPDAES*DEDEDFKEQTR.L(+4) NP_476489 -0.17 ± 0.09 PREDICTED: AHNAK nucleoprotein isoform 2 isoform 1 R.KGDRS*PEPGQTWAHEVFSSR.S(+3) XP_001078152 -0.17 ± 0.08 131 132 heat shock 105kDa K.IES*PKLER.T(+2) NP_001011901 -0.16 ± 0.07 133 BCL2-associated transcription factor 1 R.RIDIS*PSALR.K(+2) NP_001041317 -0.16 ± 0.17 134 family with sequence similarity 83, member H R.KGS*PTPAYPER.K(+2) NP_001124037 -0.16 ± 0.07 135 erythrocyte protein band 4.1-like 1 isoform L R.RLPSSPAS*PSPK.G(+2) NP_067713 -0.16 ± 0.07 136 programmed cell death 4 R.DSGRGDS*VSDNGSEAVR.S(+3) NP_071601 -0.16 ± 0.09 137 drebrin-like K.LRS*PFLQK.Q(+3) NP_112642 -0.16 ± 0.13 138 nucleobindin 1 R.AQRLS*QETEALGR.S(+2) NP_445915 -0.16 ± 0.13 139 PREDICTED: Beta-2-syntrophin isoform 2 R.LVHSGS*GCRSPSLGSDLTFATR.T(+3) XP_001071043 -0.16 ± 0.1 140 PREDICTED: Beta-2-syntrophin isoform 2 R.LVHSGSGCRS*PSLGSDLTFATR.T(+3) XP_001071043 -0.16 ± 0.1 PREDICTED: mitogen-activated protein kinase kinase kinase R.S*DSDEAAPTAPSPPPS*LLPPSPS*T*NPLVDVELE XP_001073032 -0.16 ± 0.17 141 10 SFKK.D(+4) PREDICTED: microsomal glutathione S-transferase 3 R.AKM#AVLSKEY*GFVLLTGAASFVMVLHLAINVGK.A XP_001076104 -0.16 ± 0.48 142 (+3) 143 PREDICTED: dynein heavy chain domain 3 R.YPPNAPFY*KIM#VPT*VDT*VR.Y(+3) XP_220603 -0.16 ± 0.37 144 PCTAIRE protein kinase 3 R.NEDGRDEPGQLS*PGVQYQQR.Q(+3) NP_001093976 -0.15 ± 0.04 145 catenin (cadherin associated protein), alpha 2 R.SRT*SVQTEDDQLIAGQSAR.A(+3) NP_001100068 -0.15 ± 0.08 146 catenin (cadherin associated protein), alpha 2 R.SRTS*VQTEDDQLIAGQSAR.A(+3) NP_001100068 -0.15 ± 0.08 147 catenin (cadherin associated protein), delta 1 R.VGGS*SVDLHR.F(+3) NP_001101210 -0.15 ± 0.33 branched chain keto acid dehydrogenase E1, alpha R.IGHHS*TSDDS*SAYRSVDEVNYWDKQDHPISR.L(+ NP_036914 -0.15 ± 0.13 148 polypeptide 4) 149 ankyrin 3, epithelial isoform 1 R.AEEPVS*PLTAYQK.S(+2) NP_113993 -0.15 ± 0.12 DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 R.VGWSTM#QAS*LDLGTDKFGFGFGGTGKK.S(+3) NP_445866 -0.15 ± 0.1 150

151 PREDICTED: tankyrase 1-binding protein of 182 kDa R.KEIPAS*PDRLWGSR.L(+3) XP_215763 -0.15 ± 0.07 PREDICTED: Dystroglycan precursor (Dystrophin-associated R.IVPTPTSPAIAPPTET*MAPPVR.D(+2) XP_343484 -0.15 ± 0.25 152 glycoprotein 1) 153 LIM domain 7 R.EVSRS*PDQFSDMR.I(+3) NP_001001515 -0.14 ± 0.03 154 serine/arginine repetitive matrix 1 R.KETES*EAEDDNLDDLER.H(+3) NP_001101456 -0.14 ± 0.07 solute carrier family 14 (urea transporter), member 2 isoform K.LYESELSS*PTWPSSSQDTHPALPLLEMPEEK.D(+4 NP_808877 -0.14 ± 0.9 155 2 ) translocase of outer mitochondrial membrane 70 homolog A R.AS*PALGSGPDGSGDSLEMSSLDR.A(+2) NP_997684 -0.14 ± 0.13 156 PREDICTED: Nucleophosmin K.CGSGPVHISGQHLVAVEEDAES*EDEDEEDVKLLG XP_001071367 -0.14 ± 0.31 157 MSGK.R(+4) Rho guanine nucleotide exchange factor (GEF) 12 R.VCLEDTPERTEGVQDADTQSLVGS*PSTR.G(+3) NP_001013264 -0.13 ± 0.2 158

159 nuclear cap binding protein subunit 1, 80kDa R.RKT*SDANETEDHLESLICK.V(+4) NP_001014785 -0.13 ± 0.06 160 epsin 3 R.TPVLPSGPPITDPWAPSS*PTPK.L(+2) NP_001019962 -0.13 ± 0.17 161 serine/arginine repetitive matrix 1 R.KET*ESEAEDDNLDDLER.H(+3) NP_001101456 -0.13 ± 0.07 162 family with sequence similarity 83, member H R.KGS*PTPAYPER.K(+3) NP_001124037 -0.13 ± 0.08 163 thymopoietin K.GPPDFSSDEEREPT*PVLGSGASVGR.G(+3) NP_037019 -0.13 ± 0.09 USO1 homolog, vesicle docking protein K.LKDLGHPVEEEDES*GDQEDDDDELDDGDRDQDI.- NP_062252 -0.13 ± 0.08 164 (+4) 165 sorting nexin 16 K.FT*VY*KILVKESPEESWVVFR.R(+3) NP_071625 -0.13 ± 0.31 PREDICTED: Transcription initiation factor TFIID 105 kDa K.VTVAPVKSLTQIGTTVATTAS*SASS*GQTVLENVK. XP_001073245 -0.13 ± 0.32 166 subunit (TAFII-105) (TAFII105) K(+3) 167 paxillin R.YAHQQPPS*PSPIYSSSTK.N(+3) NP_001012147 -0.12 ± 0.09 168 Structure specific recognition protein 1 K.EGINPGYDDYADS*DEDQHDAYLER.M(+3) NP_112383 -0.12 ± 0.08 169 Structure specific recognition protein 1 K.EGINPGYDDYADSDEDQHDAY*LER.M(+3) NP_112383 -0.12 ± 0.08 170 calcium regulated heat stable protein 1 R.TFS*ATVR.A(+2) NP_690003 -0.12 ± 0.2 solute carrier family 14 (urea transporter), member 2 isoform R.YKLYESELS*SPTWPSSSQDTHPALPLLEMPEEK.D NP_808877 -0.12 ± 0.35 171 2 (+4) 172 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.MYFPDVEFDIKS*PK.F(+2) XP_001078032 -0.12 ± 0.13 173 transforming acidic coiled coil 2 isoform 2 K.TPEKLDNTPAS*PPRS*PAEPSDIPIAK.G(+3) NP_001004418 -0.11 ± 0.14 174 thyroid hormone receptor associated protein 3 R.IDIS*PSTFR.K(+2) NP_001009693 -0.11 ± 0.06 serine/arginine repetitive matrix 1 K.KPPAPPSPVQS*QS*PSTNWS*PAAPAKK.A(+3) NP_001101456 -0.11 ± 0.13 175

176 lethal giant larvae homolog 2 R.VAVGCRLS*NGEAE.-(+2) NP_001121021 -0.11 ± 0.08 acidic (leucine-rich) nuclear phosphoprotein 32 family, R.NRTPS*DVKELVLDNCR.S(+3) NP_037035 -0.11 ± 0.15 177 member A 178 heterogeneous nuclear ribonucleoprotein A1 K.SES*PKEPEQLRK.L(+3) NP_058944 -0.11 ± 0.12 179 erythrocyte protein band 4.1-like 1 isoform L R.RSEAEEGEVRT*PTK.I(+3) NP_067713 -0.11 ± 0.05 nucleoporin 98kDa K.NLNNSNLFSPVNHDSEDLAS*PSEYPENGER.F(+3) NP_112336 -0.11 ± 0.41 180

181 zinc finger, RAN-binding domain containing 2 R.ENVEYIEREES*DGEYDEFGR.K(+3) NP_113804 -0.11 ± 0.18 182 alpha-spectrin 2 R.WRS*LQQLAEER.S(+3) NP_741984 -0.11 ± 0.21 183 thyroid hormone receptor associated protein 3 R.RIDIS*PSTFR.K(+2) NP_001009693 -0.1 ± 0.1 184 connector enhancer of kinase suppressor of Ras 1 K.KIPVPETPSKT*PPDS*PQR.L(+3) NP_001034100 -0.1 ± 0.21 185 adaptor-related protein complex 3, delta 1 subunit R.VDIITEEMPENALPS*DEDDKDPNDPYR.A(+3) NP_001094189 -0.1 ± 0.21 186 protein phosphatase 4, regulatory subunit 2 R.AEETETAS*SPPEKDRENR.T(+3) NP_001100083 -0.1 ± 0.15 187 erythrocyte protein band 4.9 R.KGAEEEEEEEDDDS*EEEIKAIR.E(+3) NP_001101855 -0.1 ± 0.15 188 ATP-binding cassette, sub-family F (GCN20), member 1 R.LKQLSVPAS*DEEDEVPVPVPR.G(+3) NP_001103353 -0.1 ± 0.34 USO1 homolog, vesicle docking protein K.LKDLGHPVEEEDES*GDQEDDDDELDDGDRDQDI.- NP_062252 -0.1 ± 0.12 189 (+3) PDZ and LIM domain 5 K.GEPKEVVKPVPIT*SPAVSKVT*STTNM#AYNK.V(+3 NP_445778 -0.1 ± 0.22 190 ) StAR-related lipid transfer (START) domain containing 10 R.AGGAGEGS*DDDTSLT.-(+2) NP_001013087 -0.09 ± 0.05 191

192 epsin 3 K.GKS*PS*PVELDPFGDSSPSCK.Q(+3) NP_001019962 -0.09 ± 0.16 193 Cd4 molecule R.LLQGQSLTLILDS*NPK.V(+3) NP_036837 -0.09 ± 0.33 194 crystallin, alpha B R.APS*WIDTGLSEMR.M(+2) NP_037067 -0.09 ± 0.16 myeloid/lymphoid (trithorax homolog, Drosophila); K.TQVLS*PDSLFTAK.F(+2) NP_037349 -0.09 ± 0.07 195 translocated to, 4 adenosine monophosphate deaminase 3 K.GPPSVSPAMS*PT*TPLVLGAASKPGLAPYDMPEY NP_113732 -0.09 ± 0.09 196 QR.A(+4) 197 hepatoma-derived growth factor R.RAGDMLEDS*PKRPK.E(+3) NP_446159 -0.09 ± 0.03 PREDICTED: RNA-binding protein 14 (RNA-binding motif R.TRLS*PPR.A(+2) XP_001072105 -0.09 ± 0.15 198 protein 14) eukaryotic translation initiation factor 3, subunit 9 (eta) R.AKPAAQSEEETAAS*PAAS*PTPQSAQEPSAPGKA NP_001026810 -0.08 ± 0.32 199 EAGGEQAR.H(+4) human immunodeficiency virus type I enhancer binding R.LSGLDGQRDSSSSS*S*KTR.T(+3) NP_001099221 -0.08 ± 0.07 200 protein 1 201 tight junction protein 3 K.ASPASGHQLS*DQEEADHGR.G(+3) NP_001101543 -0.08 ± 0.26 LSM14A, SCD6 homolog A R.S*SPQLDPLRKS*PTMEQAVQTASAHLPAPAPVGR. NP_001121024 -0.08 ± 0.23 202 R(+4) LSM14A, SCD6 homolog A R.SS*PQLDPLRKSPT*MEQAVQTASAHLPAPAPVGR. NP_001121024 -0.08 ± 0.23 203 R(+4) 204 parvin, alpha K.SPSVPKS*PTPKS*PPSR.K(+3) NP_065707 -0.08 ± 0.06 205 tight junction protein 2 R.RQQYS*DQEYHSSTEK.L(+3) NP_446225 -0.08 ± 0.06 206 PREDICTED: tankyrase 1-binding protein of 182 kDa K.TWVTSSADPVS*EHGVSTSPVLLAK.V(+3) XP_215763 -0.08 ± 0.16 PREDICTED: Poly(rC)-binding protein 1 (Alpha-CP1) (hnRNP- R.VMTIPYQPMPASS*PVICAGGQDR.C(+3) XP_575592 -0.08 ± 0.08 207 E1) 208 thyroid hormone receptor associated protein 3 R.ERS*PALKS*PLQSVVVR.R(+3) NP_001009693 -0.07 ± 0.1 209 leucine rich repeat (in FLII) interacting protein 1 K.AENQRPVEDSALS*PGPLAGAK.C(+3) NP_001014291 -0.07 ± 0.11 210 syntrophin, acidic 1 K.NSAGGTSVGWDS*PPAS*PLQR.Q(+2) NP_001094371 -0.07 ± 0.13 211 ladinin K.GRPEETAAQQKS*PVSEK.T(+3) NP_001101412 -0.07 ± 0.29 serine/arginine repetitive matrix 1 K.KPPAPPS*PVQS*QSPSTNWS*PAAPAKK.A(+3) NP_001101456 -0.07 ± 0.1 212

213 EPS8-like 2 R.RAEDSYSNVHTS*PEAEGAPHL.-(+3) NP_001101978 -0.07 ± 0.12 214 myosin, heavy chain 9, non-muscle R.KGTGDCS*DEEVDGKADGADAK.A(+4) NP_037326 -0.07 ± 0.13 215 splicing factor 1 isoform 2 R.TGDLGIPPNPEDRS*PS*PEPIYNSEGK.R(+3) NP_478117 -0.07 ± 0.13 216 calnexin K.QKSDAEEDGGT*GSQDEEDSKPK.A(+3) NP_742005 -0.07 ± 0.62 217 calnexin K.QKSDAEEDGGTGS*QDEEDSKPK.A(+3) NP_742005 -0.07 ± 0.62 218 PREDICTED: (PDHE1-A type I) isoform 1 R.YHGHS*MSDPGVS*YR.T(+2) XP_001060860 -0.07 ± 0.18 PREDICTED: RAB11 family interacting protein 5 (class I) R.TYS*DEASQLR.A(+2) XP_001073801 -0.07 ± 0.08 219 isoform 1 220 PREDICTED: myosin XVIIIa K.SLAPDLS*DDEHDPVDSISRPR.F(+3) XP_001080824 -0.07 ± 0.09 221 PREDICTED: tankyrase 1-binding protein of 182 kDa K.TWVTSSADPVSEHGVS*TSPVLLAK.V(+3) XP_215763 -0.07 ± 0.16 222 PREDICTED: tankyrase 1-binding protein of 182 kDa K.TWVTSSADPVSEHGVST*SPVLLAK.V(+3) XP_215763 -0.07 ± 0.16 223 PREDICTED: tankyrase 1-binding protein of 182 kDa K.TWVTSSADPVSEHGVSTS*PVLLAK.V(+3) XP_215763 -0.07 ± 0.16 histone deacetylase 1 R.MLPHAPGVQMQAIPEDAIPEES*GDEDEEDPDKR.I( NP_001020580 -0.06 ± 0.11 224 +4) 225 syntrophin, acidic 1 K.NSAGGTSVGWDS*PPAS*PLQR.Q(+3) NP_001094371 -0.06 ± 0.12 EF hand domain family, member B R.APLGKSHDQTPGLPKGMDVINTT*LGTPT*IR.E(+3) NP_001100349 -0.06 ± 0.3 226 ladinin R.SVERLPS*VEEAEVSKPS*PPASKDEGEEFQAILR.T NP_001101412 -0.06 ± 0.17 227 (+4) 228 erythrocyte protein band 4.9 K.ST*SPPPS*PEVWAESR.T(+2) NP_001101855 -0.06 ± 0.07 PREDICTED: OTU domain containing 4 protein isoform 1 R.LQRPKEES*S*EDENEVSNILR.S(+3) XP_001074219 -0.06 ± 0.23 229 PREDICTED: U2 (RNU2) small nuclear RNA auxiliary factor 2 R.GAKEEHGGLIRS*PR.H(+3) XP_218195 -0.06 ± 0.13 230 isoform b 231 protein kinase, cAMP dependent regulatory, type II beta R.RAS*VCAEAYNPDEEEDDAESR.I(+3) NP_001025191 -0.05 ± 0.18 232 non-SMC condensin II complex, subunit D3 R.T*Y*AAQS*LVQLLS*KLPSK.E(+3) NP_001029172 -0.05 ± 0.46 233 utrophin K.NVRPQPPT*SPEGR.T(+2) NP_037202 -0.05 ± 0.05 234 utrophin K.NVRPQPPTS*PEGR.T(+2) NP_037202 -0.05 ± 0.05 235 protein kinase, cAMP dependent regulatory, type I, alpha R.TDSREDEIS*PPPPNPVVK.G(+3) NP_037313 -0.05 ± 0.13 small glutamine-rich tetratricopeptide repeat (TPR)- R.GPDRT*PPSEEDSAEAER.L(+3) NP_073194 -0.05 ± 0.12 236 containing, alpha 237 myosin, heavy chain 10, non-muscle R.QLHIEGASLELS*DDDTESK.T(+3) NP_113708 -0.05 ± 0.46 238 chloride channel, nucleotide-sensitive, 1A R.FGEESKEPFS*DEDEDDNDDVEPISEFR.F(+3) NP_113907 -0.05 ± 0.44 239 septin 2 K.IYHLPDAES*DEDEDFKEQTR.L(+3) NP_476489 -0.05 ± 0.02 240 PREDICTED: topoisomerase (DNA) II beta K.KTSFDQDS*DVDIFPSDFTSEPPALPR.T(+3) XP_001067768 -0.05 ± 0.24 PREDICTED: Epithelial protein lost in neoplasm (mEPLIN) R.SEAQQPIYTKPLS*PDAR.T(+3) XP_217039 -0.05 ± 0.08 241

242 proteasome subunit alpha type 3-like K.ESLKEEDES*DDDNM.-(+2) NP_001004094 -0.04 ± 0.04 eukaryotic translation elongation factor 1 delta K.GATPAEDDEDNDIDLFGS*DEEEEDKEAAR.L(+3) NP_001013122 -0.04 ± 0.11 243 catenin (cadherin associated protein), delta 1 R.LRS*YEDMIGEEVPPDQYYWAPLAQHER.G(+3) NP_001101210 -0.04 ± 0.29 244

245 catenin (cadherin associated protein), delta 1 R.VGGS*SVDLHR.F(+2) NP_001101210 -0.04 ± 0.27 246 golgi autoantigen, golgin subfamily a, 3 K.DKFMLQAKVS*ELK.N(+3) NP_001101317 -0.04 ± 0.41 247 serine/arginine repetitive matrix 1 R.RYS*PPIQR.R(+2) NP_001101456 -0.04 ± 0.05 248 EPS8-like 2 K.YWGPAS*PTHK.L(+2) NP_001101978 -0.04 ± 0.29 249 EPS8-like 2 K.YWGPASPT*HK.L(+2) NP_001101978 -0.04 ± 0.29 250 phosphoglucomutase 1 K.AIGGIILTAS*HNPGGPNGDFGIK.F(+3) NP_058729 -0.04 ± 0.16 251 reticulon 4 R.GPLPAAPPAAPERQPS*WER.S(+3) NP_114019 -0.04 ± 0.05 252 tripartite motif-containing 28 R.SRS*GEGEVSGLMR.K(+2) NP_446368 -0.04 ± 0.06 253 calnexin K.QKS*DAEEDGGTGSQDEEDSKPK.A(+3) NP_742005 -0.04 ± 0.6 254 progesterone receptor membrane component 2 R.LLKPGEEPS*EYTDEEDTKDHSKQD.-(+3) NP_001008375 -0.03 ± 0.11 255 phosphoglucomutase 1 K.AIGGIILT*ASHNPGGPNGDFGIK.F(+3) NP_058729 -0.03 ± 0.15 256 heat shock protein 1 R.SPS*WEPFR.D(+2) NP_114176 -0.03 ± 0.15 257 NSFL1 (p97) cofactor (p47) R.KKS*PNELVDDLFK.G(+3) NP_114187 -0.03 ± 0.06 258 p21-activated kinase 2 K.YLS*FTPPEKDGFPSGTPALNTK.G(+3) NP_445758 -0.03 ± 0.21 259 PREDICTED: tankyrase 1-binding protein of 182 kDa K.SSGSLS*PGLETEDPLEAR.E(+2) XP_215763 -0.03 ± 0.05 260 PREDICTED: tetracycline transporter-like protein R.AS*S*VTLGFHT*AANLLS*PLALLR.F(+3) XP_223533 -0.03 ± 0.24 hypothetical protein LOC287534 K.KKGDGVLDS*PDSGLPPS*PSPSHWGLAAATGGG NP_001007612 -0.02 ± 0.15 261 GER.A(+4) 262 RNA binding motif protein 39 R.YRS*PYSGPK.F(+2) NP_001013225 -0.02 ± 0.08 263 WD repeat domain 42A R.VHGHS*DEEEEEEQPR.H(+3) NP_001014253 -0.02 ± 0.33 264 nuclear cap binding protein subunit 1, 80kDa R.KTS*DANETEDHLESLICK.V(+3) NP_001014785 -0.02 ± 0.16 265 leucine rich repeat containing 47 R.RES*GEGEEEVADSAR.L(+3) NP_001129138 -0.02 ± 0.16 266 phosphatidylethanolamine binding protein K.VLTPTQVMNRPS*SISWDGLDPGK.L(+3) NP_058932 -0.02 ± 0.25 267 heat shock protein 1 R.SPS*WEPFRDWYPAHSR.L(+4) NP_114176 -0.02 ± 0.25 268 hepatoma-derived growth factor R.AGDMLEDS*PKRPK.E(+3) NP_446159 -0.02 ± 0.06 269 kinesin 13B R.LEVTS*DSEDASEVPEWLR.E(+2) NP_998791 -0.02 ± 0.19 270 PREDICTED: SAPS domain family, member 3 R.IQQFDDGGS*DEEDIWEEK.H(+2) XP_001067905 -0.02 ± 0.16 PREDICTED: AHNAK nucleoprotein isoform 2 isoform 1 R.KGDRS*PEPGQTWAHEVFSSR.S(+4) XP_001078152 -0.02 ± 0.08 271

272 estrogen receptor binding site associated, antigen, 9 R.KLS*GDQITLPTTVDYSSVPK.Q(+3) NP_001009665 -0.01 ± 0.07 273 estrogen receptor binding site associated, antigen, 9 R.KLSGDQIT*LPTTVDYSSVPK.Q(+3) NP_001009665 -0.01 ± 0.07 eukaryotic translation elongation factor 1 delta K.KGATPAEDDEDNDIDLFGS*DEEEEDKEAAR.L(+3) NP_001013122 -0.01 ± 0.07 274 elongation factor Tu GTP binding domain containing 1 R.LM#AAMYTCDIMATSDVLGRVYAVLS*K.R(+3) NP_001101004 -0.01 ± 0.38 275

276 kinase insert domain protein receptor K.NKTVST*LVIQAAYVS*ALYK.C(+3) NP_037194 -0.01 ± 0.07 277 hepatoma-derived growth factor K.GNAEGS*S*DEEGKLVIDEPAKEK.N(+3) NP_446159 -0.01 ± 0.24 278 mitogen-activated protein kinase 1 R.VADPDHDHTGFLTEY*VATR.W(+3) NP_446294 -0.01 ± 0.08 heterogeneous nuclear ribonucleoprotein K K.IIPTLEEGLQLPS*PTATSQLPLESDAVECLNYQHYK NP_476482 -0.01 ± 0.08 279 .G(+4) 280 dynein cytoplasmic 1 light intermediate chain 1 R.DFQEYVEPGEDFPAS*PQRR.A(+3) NP_665715 -0.01 ± 0.04 solute carrier family 14 (urea transporter), member 2 isoform K.EMSDNNRS*PLLPEPLSSR.Y(+3) NP_808877 -0.01 ± 0.21 281 2 282 kinesin 13B R.LEVTSDS*EDASEVPEWLR.E(+2) NP_998791 -0.01 ± 0.2 283 PREDICTED: nuclear mitotic apparatus protein 1 R.LGS*PDDSNSALLSLPGYRPTTR.S(+3) XP_218972 -0.01 ± 0.12 284 nucleobindin 1 K.SGKLS*QELDFVSHNVR.T(+3) NP_445915 0 ± 0.09 285 PREDICTED: ribosomal protein L27a K.LWT*LVSKQTLVSAAKNK.T(+3) XP_001055169 0 ± 0.75 286 PREDICTED: (PDHE1-A type I) isoform 1 R.YGMGTS*VER.A(+2) XP_001060860 0 ± 0.02 287 tight junction protein 1 K.FNHNLLPSETVHKPELSS*KPPPSPK.T(+4) NP_001099736 0 ± 0.13 288 tight junction protein 1 K.FNHNLLPSETVHKPELSSKPPPS*PK.T(+4) NP_001099736 0 ± 0.13 289 catenin (cadherin associated protein), delta 1 R.GSLAS*LDSLRK.G(+2) NP_001101210 0 ± 0.25 290 synapse associated protein 1 K.SSSRDDNLPLTEAVRPKT*PPVVIK.S(+4) NP_001004253 0.01 ± 0.27 291 thyroid hormone receptor associated protein 3 K.HGLTHEELKS*PREPGYK.A(+4) NP_001009693 0.01 ± 0.11 paxillin K.TGSSS*PPGGLSKPGSQLDSMLGSLQSDLNK.L(+3) NP_001012147 0.01 ± 0.3 292 eukaryotic translation elongation factor 1 delta K.KGATPAEDDEDNDIDLFGS*DEEEEDKEAAR.L(+4) NP_001013122 0.01 ± 0.14 293 high-mobility group nucleosome binding domain 1 K.QAEVADQQTTDLPAENGETENQS*PAS*EEEKEAK. NP_001013202 0.01 ± 0.24 294 S(+3) 295 ankyrin 3, epithelial isoform 2 R.RQS*FTSLALR.K(+2) NP_001029156 0.01 ± 0.22 oxysterol binding protein K.GDMS*DEDDENEFFDAPEIITMPENLGHK.R(+3) NP_001102397 0.01 ± 0.29 296

297 hypothetical protein LOC500552 K.KLQLERPVS*PEAQADLQR.N(+3) NP_001128101 0.01 ± 0.11 298 leucine rich repeat containing 47 R.RES*GEGEEEVADSAR.L(+2) NP_001129138 0.01 ± 0.09 heterogeneous nuclear ribonucleoprotein U R.AKS*PQPPVEEEDEHFDDTVVCLDTYNCDLHFK.I(+ NP_476480 0.01 ± 0.1 299 4) 300 numb homolog R.RTPS*EADRWLEEVSK.S(+3) NP_579821 0.01 ± 0.07 solute carrier family 14 (urea transporter), member 2 isoform K.EMS*DNNRSPLLPEPLSSR.Y(+3) NP_808877 0.01 ± 0.22 301 2 302 zinc finger, CCCH-type with G patch domain R.ITDVDNGYY*TVK.F(+2) NP_001009656 0.02 ± 0.13 303 BCL2-associated transcription factor 1 K.LKELFDYS*PPLHK.S(+3) NP_001041317 0.02 ± 0.12 304 cingulin-like 1 R.NCFPKPCGS*QPNS*PTPEDLAK.T(+3) NP_001101634 0.02 ± 0.08 305 utrophin K.NVRPQPPTS*PEGR.T(+3) NP_037202 0.02 ± 0.03 histone deacetylase 2 R.MLPHAPGVQMQAIPEDAVHEDS*GDEDGEDPDKR. NP_445899 0.02 ± 0.13 306 I(+4) PREDICTED: Beta-2-syntrophin isoform 2 R.GLGPPS*PPAPPRGPAGEASAS*PPVRR.V(+3) XP_001071043 0.02 ± 0.14 307

308 PREDICTED: Myocyte-specific enhancer factor 2B R.NLRTQVPVLGNLAS*SS*RGSAVHPPHR.T(+3) XP_574970 0.02 ± 0.65 309 LIM domain 7 R.SAS*VNKEPICLTGIMR.R(+3) NP_001001515 0.03 ± 0.02 paxillin K.T*GSSSPPGGLSKPGSQLDSMLGSLQSDLNK.L(+3) NP_001012147 0.03 ± 0.31 310 spectrin, beta, non-erythrocytic 1 K.VSEEAESQQWDTSKGDQVSQNGLPAEQGS*PR.M NP_001013148 0.03 ± 0.28 311 (+3) B-cell linker R.FTEGGS*PAADGPVPSFPFNSTFADQEAELHGKP NP_001020938 0.03 ± 0.14 312 WYAGACDR.K(+4) eukaryotic translation initiation factor 3, subunit 9 (eta) R.AKPAAQSEEETAAS*PAAS*PTPQSAQEPSAPGK.A NP_001026810 0.03 ± 0.24 313 (+3) 314 myosin, heavy chain 9, non-muscle K.GTGDCS*DEEVDGKADGADAK.A(+3) NP_037326 0.03 ± 0.06 315 carnitine palmitoyltransferase 1B R.SPIM#VDSNY*Y*AMDFVLIKNTSQQAAR.L(+3) NP_037332 0.03 ± 0.12 adducin 1 (alpha) K.SPPDQSAVPNT*PPS*TPVKLEGGLPQEPTSR.D(+3 NP_058686 0.03 ± 0.09 316 ) adducin 1 (alpha) K.SPPDQSAVPNT*PPST*PVKLEGGLPQEPTSR.D(+3 NP_058686 0.03 ± 0.09 317 ) 318 matrin 3 R.RDS*FDDRGPSLNPVLDYDHGSR.S(+4) NP_062022 0.03 ± 0.09 319 PREDICTED: DNA2 DNA replication helicase 2-like K.DY*TLIVGM#PGT*GKTTT*ICALVR.I(+3) XP_001080557 0.03 ± 0.12 320 phosphoprotein enriched in astrocytes 15A K.YKDIIRQPS*EEEIIK.L(+3) NP_001013249 0.04 ± 0.14 chromobox homolog 5 (HP1 alpha homolog, Drosophila) R.KSS*FSNSADDIK.S(+2) NP_001100267 0.04 ± 0.09 321

322 serine/arginine repetitive matrix 1 R.RLS*PS*ASPPR.R(+2) NP_001101456 0.04 ± 0.07 DNA segment, Chr 4, ERATO Doi 22, expressed isoform 1 R.ILGSAS*PEEEQEKPILDRPTR.I(+3) NP_001108071 0.04 ± 0.11 323 myeloid/lymphoid or mixed-lineage leukemia (trithorax K.ITSVS*TGNLCTEEQT*PPPRPEAYPIPTQTYTR.E(+ NP_037349 0.04 ± 0.25 324 homolog, Drosophila); translocated to, 4 3) PREDICTED: pleckstrin homology domain containing, family R.YIDLEPVAPLS*PEELKEK.Q(+3) XP_001060716 0.04 ± 0.16 325 A member 6 326 PREDICTED: PDHE1-A type I isoform 1 R.YHGHS*MSDPGVS*YR.T(+3) XP_001060860 0.04 ± 0.14 tight junction protein 1 K.VQIPVSHPDPDPVS*DNEDDSY*DEDVHDPR.S(+3) NP_001099736 0.05 ± 0.06 327

328 serine/arginine repetitive matrix 1 R.RRS*PS*PAPPPPPPPPPR.R(+3) NP_001101456 0.05 ± 0.09 calnexin K.QKSDAEEDGGTGS*QDEEDSKPKAEEDEILNR.S(+ NP_742005 0.05 ± 0.15 329 4) PREDICTED: TBC1 domain family member 4 (Akt substrate R.HAS*APSHVQPSDSEK.N(+3) XP_001074155 0.05 ± 0.09 330 of 160 kDa) (AS160) isoform 1 PREDICTED: splicing factor, arginine/serine-rich 1 (ASF/SF2) R.VKVDGPRS*PS*YGR.S(+3) XP_001081187 0.05 ± 0.11 331

332 PREDICTED: tankyrase 1-binding protein of 182 kDa R.NMAPGAGCS*PGEPR.E(+2) XP_215763 0.05 ± 0.17 333 PRP4 pre-mRNA processing factor 4 homolog B K.LCDFGSASHVADNDITPY*LVSR.F(+3) NP_001011923 0.06 ± 0.08 334 cingulin-like 1 R.S*RSVDSAFPFGLQGNTEYLTEFSR.N(+3) NP_001101634 0.06 ± 0.38 335 plectin 1 R.SSS*VGSSSSYPISSAVPR.T(+2) NP_071796 0.06 ± 0.11 calpastatin isoform a K.LLEKNEAITGPLPDS*PKPMGIDHAIDALSSDFTCSS NP_445747 0.06 ± 0.27 336 *PTGK.Q(+4) 337 hepatoma-derived growth factor-related protein 2 R.KRS*EGLSLDR.K(+3) NP_598232 0.06 ± 0.1 338 kinesin 13B R.RRS*SGLQPQGAPEAR.R(+3) NP_998791 0.06 ± 0.52 339 PREDICTED: hypothetical protein R.RDT*ATEIQDVGVPR.N(+3) XP_001077427 0.06 ± 0.07 340 PREDICTED: tensin R.QGSPT*PALPEKR.R(+3) XP_237286 0.06 ± 0.12 341 BCL2-associated transcription factor 1 K.KAEGEPQEES*PLKSK.S(+3) NP_001041317 0.07 ± 0.08 tight junction protein 1 K.VQIPVSHPDPDPVS*DNEDDS*YDEDVHDPR.S(+4) NP_001099736 0.07 ± 0.1 342

343 NCK adaptor protein 1 K.RKPS*VPDTASPADDSFVDPGER.L(+3) NP_001100321 0.07 ± 0.09 344 hypothetical protein LOC308341 K.NAKRPT*TQPPGIPIT*KRPR.V(+3) NP_001128017 0.07 ± 0.15 myeloid/lymphoid or mixed-lineage leukemia (trithorax K.ITSVST*GNLCTEEQT*PPPRPEAYPIPTQTYTR.E(+ NP_037349 0.07 ± 0.24 345 homolog, Drosophila); translocated to, 4 3) 346 plectin 1 R.SS*SVGSSSSYPISSAVPR.T(+2) NP_071796 0.07 ± 0.11 347 heat shock protein 1 R.SPS*WEPFRDWYPAHSR.L(+3) NP_114176 0.07 ± 0.13 348 PREDICTED: vacuolar protein sorting 13B isoform 1 K.CT*CT*IFM#AEFNLLDRLLPVIMGGK.N(+3) XP_001058951 0.07 ± 0.37 ladinin R.SVERLPS*VEEAEVSKPSPPASKDEGEEFQAILR.T( NP_001101412 0.08 ± 0.21 349 +4) protein phosphatase 1, regulatory subunit 2 K.IDEPDTPYHNMIGDDEDVCS*DSEGNEVMTPEILAK NP_620178 0.08 ± 0.09 350 .K(+3) 351 PREDICTED: hypothetical protein isoform 2 K.LGGNEALS*PTS*PSKESRPGEWR.T(+3) XP_001065403 0.08 ± 0.16 PREDICTED: Septin-4 (Peanut-like protein 2) (Brain protein K.IYQFPDCDS*DEDEDFKLQDQALK.E(+3) XP_001081164 0.08 ± 0.11 352 H5) 353 spectrin, beta, non-erythrocytic 1 R.T*SSKESS*PVPS*PTSDRK.A(+3) NP_001013148 0.09 ± 0.15 354 spectrin, beta, non-erythrocytic 1 R.TSS*KES*SPVPS*PTSDRK.A(+3) NP_001013148 0.09 ± 0.15 355 spectrin, beta, non-erythrocytic 1 R.TSS*KESS*PVPS*PTSDRK.A(+3) NP_001013148 0.09 ± 0.15 356 catenin (cadherin associated protein), delta 1 R.GSLAS*LDS*LRK.G(+2) NP_001101210 0.09 ± 0.28 357 PREDICTED: hypothetical protein isoform 2 K.LGGNEALSPT*S*PSKESRPGEWR.T(+3) XP_001065403 0.09 ± 0.16 PREDICTED: Semaphorin-4B precursor (Semaphorin C) R.VRLGS*EIRDSVV.-(+2) XP_001065984 0.09 ± 0.23 358 (Sema C) 359 progesterone receptor membrane component 2 R.LLKPGEEPSEYT*DEEDTKDHSKQD.-(+4) NP_001008375 0.1 ± 0.11 360 protein phosphatase 1, regulatory (inhibitor) subunit 12B R.RLS*GTSDIEEKENR.E(+3) NP_001100648 0.1 ± 0.09 361 pinin, desmosome associated protein R.RGFS*DSGGGPPAK.Q(+2) NP_001102493 0.1 ± 0.16 362 ATP-binding cassette, sub-family F (GCN20), member 1 K.GAEQGS*EEEKEEKEGEVK.A(+3) NP_001103353 0.1 ± 0.14 363 phosphatidylethanolamine binding protein K.VLTPTQVMNRPSS*ISWDGLDPGK.L(+3) NP_058932 0.1 ± 0.18 signal transducer and activator of transcription 1 isoform R.LQSTENLLPMS*PEEFDEMSK.I(+2) NP_116001 0.1 ± 0.24 364 alpha 365 zyxin R.S*PGGPGPLTLK.E(+2) NP_446213 0.1 ± 0.43 366 protein phosphatase 1, regulatory (inhibitor) subunit 12A R.RLGS*TSDIEEKENR.E(+3) NP_446342 0.1 ± 0.09 phosphoribosyl pyrophosphate synthetase-associated protein R.LGIAVIHGEAQDAESDLVDGRHS*PPMVR.S(+4) NP_476472 0.1 ± 0.07 367 2 PREDICTED: Secretory carrier-associated membrane protein R.EPPPAY*EPPAPAPAPLPPPS*APS*VQSS*RK.L(+3 XP_342280 0.1 ± 0.19 368 3 ) solute carrier family 14 (urea transporter), member 2 isoform K.LYESELS*SPTWPSSSQDTHPALPLLEMPEEK.D(+3 NP_808877 0.11 ± 0.24 369 2 ) BTB (POZ) domain containing 7 R.EILS*SLLPFVRIEHILPMNS*EVLSDAM#KR.G(+3) NP_001102190 0.11 ± 0.33 370 BTB (POZ) domain containing 7 R.EILSS*LLPFVRIEHILPMNS*EVLSDAM#KR.G(+3) NP_001102190 0.11 ± 0.33 371

372 myosin, heavy chain 9, non-muscle R.KGTGDCS*DEEVDGKADGADAK.A(+3) NP_037326 0.11 ± 0.07 373 heterogeneous nuclear ribonucleoprotein A1 K.SES*PKEPEQLR.K(+2) NP_058944 0.11 ± 0.32 374 heterogeneous nuclear ribonucleoprotein D isoform a K.IDASKNEEDEGHSNSS*PR.H(+3) NP_077380 0.11 ± 0.23 375 PREDICTED: PDHE1-A type I isoform 1 R.YHGHS*M#SDPGVSYR.T(+3) XP_001060860 0.11 ± 0.17 376 related RAS viral (r-ras) oncogene homolog 2 R.KFQEQECPPS*PEPTRK.E(+3) NP_001013452 0.12 ± 0.06 377 BCL2-associated transcription factor 1 K.GRTDGDWDDQEVLDYFS*DKESAK.Q(+3) NP_001041317 0.12 ± 0.13 eukaryotic translation elongation factor 1 beta 2 K.YGPVSVADTTGSGAADAKDDDDIDLFGS*DDEEES NP_001102269 0.12 ± 0.17 378 EDAKR.L(+4) hypothetical protein LOC500552 K.ASSLAASESPGNALPTGAPETEPRS*PQS*PASK.A( NP_001128101 0.12 ± 0.13 379 +3) 380 mitogen activated protein kinase 3 R.IADPEHDHTGFLT*EYVATR.W(+3) NP_059043 0.12 ± 0.06 381 mitogen activated protein kinase 3 R.IADPEHDHTGFLTEY*VATR.W(+3) NP_059043 0.12 ± 0.06 cortactin isoform B R.AKKQT*PPAS*PSPQPAEDRPPSS*PIYEDAAPLK.A NP_068640 0.12 ± 0.17 382 (+4) cortactin isoform B R.AKKQT*PPAS*PSPQPAEDRPPSSPIY*EDAAPLK.A NP_068640 0.12 ± 0.17 383 (+4) 384 cysteine string protein R.S*LSTSGESLYHVLGLDK.N(+2) NP_077075 0.12 ± 0.2 385 heterogeneous nuclear ribonucleoprotein D isoform a K.IDASKNEEDEGHSNS*SPR.H(+3) NP_077380 0.12 ± 0.23 386 spectrin, beta, non-erythrocytic 1 R.TSS*KESSPVPS*PTSDRK.A(+3) NP_001013148 0.13 ± 0.22 histone deacetylase 1 R.MLPHAPGVQMQAIPEDAIPEES*GDEDEEDPDKR.I( NP_001020580 0.13 ± 0.14 387 +3) coiled-coil domain containing 43 K.AALLAQYADVT*DEEDEADEKADPGASTANIGSDK. NP_001094198 0.13 ± 0.15 388 S(+3) tight junction protein 1 K.KVQIPVSHPDPDPVS*DNEDDS*YDEDVHDPR.S(+4 NP_001099736 0.13 ± 0.11 389 ) tight junction protein 1 K.KVQIPVSHPDPDPVS*DNEDDSY*DEDVHDPR.S(+4 NP_001099736 0.13 ± 0.11 390 ) 391 Cdc42-binding protein kinase beta K.HSTPSNSSNPSGPPS*PNS*PHR.S(+3) NP_446072 0.13 ± 0.33 heterogeneous nuclear ribonucleoprotein K K.IIPTLEEGLQLPS*PTATSQLPLESDAVECLNYQHYK NP_476482 0.13 ± 0.11 392 .G(+3) PREDICTED: FCH domain only 2 K.LSGINEIPRPFS*PPITS*NTSPPPTAPLAR.A(+3) XP_219503 0.13 ± 0.13 393 PREDICTED: Apoptotic chromatin condensation inducer in K.SSSFSEEKGES*DDEKPR.K(+3) XP_240178 0.13 ± 0.08 394 the nucleus (Acinus) 395 RNA binding motif protein 17 R.RPDPDS*DEDEDYERER.R(+3) NP_001013076 0.14 ± 0.14 396 chordin-like 2 R.HFQAIGMGGT*TIKIILK.E(+2) NP_001101007 0.14 ± 0.66 397 receptor (chemosensory) transporter protein 4 R.SPSPSPSPSPKSHS*SSPS*R.S(+3) NP_001101791 0.14 ± 0.25 398 aquaporin 2 (collecting duct) R.RQS*VELHSPQSLPR.G(+3) NP_037041 0.14 ± 0.35 PREDICTED: caspase recruitment domain family, member 14 R.LQGATLDDTDT*DLEFEMLDGADLS*QT*EDSLQGS XP_001081765 0.14 ± 0.12 399 SR.S(+4) PREDICTED: FCH domain only 2 K.LSGINEIPRPFS*PPITSNTS*PPPTAPLAR.A(+3) XP_219503 0.14 ± 0.13 400 nucleosome assembly protein 1-like 4 R.EFITGDVEPTDAESAWHS*ENEEDDKLAGDMK.N(+ NP_001012170 0.15 ± 0.16 401 3) aquaporin 4 isoform 1 K.GSYMEVEDNRS*QVETEDLILKPGVVHVIDIDRGDE NP_036957 0.15 ± 0.12 402 K.K(+4) 403 PREDICTED: serine/arginine repetitive matrix 2 R.S*RTPPSAPSQSR.M(+3) XP_001056343 0.15 ± 0.19 actin-binding LIM protein 1 K.LRPT*RTSSESIYSRPGSSIPGS*PGHTIYAK.V(+4) NP_001037859 0.16 ± 0.17 404

405 death-associated protein K.DKDDQEWESTS*PPKPTVYISGVIAR.G(+3) NP_071971 0.16 ± 0.15 calpastatin isoform a K.NEAITGPLPDS*PKPMGIDHAIDALSSDFTCSS*PTG NP_445747 0.16 ± 0.43 406 K.Q(+4) 407 PREDICTED: PDHE1-A type I isoform 1 R.YHGHSMS*DPGVSYR.T(+3) XP_001060860 0.16 ± 0.08 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 R.NRS*NS*FSDEREFSAPST*PTGTLEFAGGEGK.G(+ XP_001078032 0.16 ± 0.33 408 3) PREDICTED: Epithelial protein lost in neoplasm (mEPLIN) K.DLWASKGENEETLGRPAQPPSAGETPHS*PGVED XP_217039 0.16 ± 0.3 409 APIAK.V(+4) hypothetical protein LOC296179 K.NSKEPVIEDILNLESSSTSSGS*PVS*PGLYSK.T(+3) NP_001099985 0.18 ± 0.47 410

411 hypothetical protein LOC300783 K.RPLFLAPDFDRWLDES*DAEMELR.A(+4) NP_001100301 0.18 ± 0.36 hypothetical protein LOC500552 R.LKASSLAASESPGNALPTGAPETEPRS*PQS*PASK NP_001128101 0.18 ± 0.32 412 .A(+3) cortactin isoform B K.KQT*PPAS*PSPQPAEDRPPSS*PIYEDAAPLK.A(+4 NP_068640 0.18 ± 0.3 413 ) 414 heterogeneous nuclear ribonucleoprotein D isoform a K.IDASKNEEDEGHSNS*S*PR.H(+3) NP_077380 0.18 ± 0.43 415 PREDICTED: THUMP domain containing 1 K.FIDKDQQPS*GS*EGEDDDAEAALKK.E(+3) XP_001079074 0.18 ± 0.19 SWI/SNF related, matrix associated, actin dependent R.KHS*PS*PPPPTATESR.K(+3) NP_001100331 0.19 ± 0.13 416 regulator of chromatin, subfamily c, member 1 417 heat shock protein 1 R.SPS*WEPFRDWYPAHSR.L(+2) NP_114176 0.19 ± 1.35 418 PREDICTED: serine/arginine repetitive matrix 2 R.RQPS*PQPS*PRDQQSSER.V(+3) XP_001056343 0.19 ± 0.27 419 Rho GTPase activating protein 1 K.SSS*PEPVTHLKWDDPYYDIAR.H(+3) NP_001101217 0.2 ± 0.46 420 peroxisome biogenesis factor 1 K.EQGRT*VFVLSPILLQK.I(+2) NP_001102690 0.2 ± 0.59 421 PC4 and SFRS1 interacting protein 1 K.NLAKPGVT*S*TSDSEEDDDQEGEKKR.K(+3) NP_786941 0.22 ± 0.27 422 PC4 and SFRS1 interacting protein 1 K.NLAKPGVTS*T*SDSEEDDDQEGEKKR.K(+3) NP_786941 0.22 ± 0.27 423 serine/arginine repetitive matrix 1 R.RYS*PS*PPPK.R(+2) NP_001101456 0.23 ± 0.15 eukaryotic translation elongation factor 1 beta 2 K.YGPVSVADTTGSGAADAKDDDDIDLFGS*DDEEES NP_001102269 0.23 ± 0.26 424 EDAKR.L(+3) 425 cysteine string protein R.SLS*TSGESLYHVLGLDK.N(+2) NP_077075 0.23 ± 0.16 426 coiled-coil domain containing 105 K.FNQEM#Y*VTRGLIK.G(+3) NP_001020945 0.24 ± 0.26 FERM, RhoGEF (Arhgef) and pleckstrin domain protein 1 R.LGAPENSGIST*LER.G(+2) NP_001100757 0.24 ± 0.22 427 (chondrocyte-derived) myosin light chain, regulatory B R.ATS*NVFAMFDQSQIQEFKEAFNMIDQNR.D(+3) NP_059039 0.24 ± 0.44 428

429 phosphatidylinositol 4-kinase type 2 alpha R.VAAAGS*GPSPPCS*PGHDRER.Q(+3) NP_446187 0.24 ± 0.16 430 phosphatidylinositol 4-kinase type 2 alpha R.VAAAGSGPS*PPCS*PGHDRER.Q(+3) NP_446187 0.24 ± 0.16 431 myosin phosphatase-Rho interacting protein isoform 1 R.MDIDRS*PGLLGTPDLK.T(+3) NP_446266 0.24 ± 0.12 432 estrogen receptor binding site associated, antigen, 9 R.KLS*GDQITLPTTVDYSSVPK.Q(+2) NP_001009665 0.25 ± 0.2 433 olfactory receptor Olr611 R.LIS*S*K.L(+1) NP_001000926 0.26 ± 0.35 catenin (cadherin associated protein), delta 1 R.FHPEPYGLEDDQRS*MGYDDLDYGMMSDYGTAR. NP_001101210 0.26 ± 0.15 434 R(+4) 435 myosin light chain, regulatory B R.AT*SNVFAMFDQSQIQEFK.E(+3) NP_059039 0.26 ± 0.34 436 myosin light chain, regulatory B R.ATS*NVFAMFDQSQIQEFK.E(+3) NP_059039 0.26 ± 0.34 437 myosin light chain, regulatory B R.AT*SNVFAMFDQSQIQEFK.E(+2) NP_059039 0.27 ± 0.25 438 myosin light chain, regulatory B R.ATS*NVFAMFDQSQIQEFK.E(+2) NP_059039 0.27 ± 0.25 hypothetical protein LOC296179 K.NSKEPVIEDILNLESSSTSSGSPVS*PGLYSK.T(+3) NP_001099985 0.28 ± 0.16 439 chromobox homolog 5 (HP1 alpha homolog, Drosophila) R.KSSFS*NSADDIKSK.K(+3) NP_001100267 0.28 ± 0.73 440

441 high mobility group AT-hook 1 K.KLEKEEEEGISQES*S*EEEQ.-(+3) NP_647543 0.28 ± 0.33 442 PREDICTED: tankyrase 1-binding protein of 182 kDa R.ASRVPS*S*DEEVVEEPQSR.R(+3) XP_215763 0.28 ± 0.33 cortactin isoform B K.KQT*PPAS*PSPQPAEDRPPS*SPIYEDAAPLK.A(+3 NP_068640 0.29 ± 0.27 443 ) nuclear casein kinase and cyclin-dependent kinase substrate K.VVDYSQFQES*DDADEDYGRDSGPPAKK.I(+3) NP_073636 0.29 ± 0.37 444 1 nuclear casein kinase and cyclin-dependent kinase substrate R.KVVDYSQFQES*DDADEDYGRDSGPPAK.K(+3) NP_073636 0.29 ± 0.37 445 1 Structure specific recognition protein 1 R.GLKEGINPGYDDYADS*DEDQHDAYLER.M(+4) NP_112383 0.29 ± 0.86 446 solute carrier family 14 (urea transporter), member 2 isoform R.YKLYESELSS*PTWPSSSQDTHPALPLLEMPEEKD NP_808877 0.29 ± 0.67 447 2 LR.S(+4) 448 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 K.GDVDVSIPNVEGDLQGPS*LDIKSPK.L(+3) XP_001078032 0.29 ± 0.42 PREDICTED: SH2 domain binding protein 1 (tetratricopeptide R.RRPPKGEEGS*DDDETENGPKPK.K(+4) XP_238127 0.29 ± 0.14 449 repeat containing) PREDICTED: E3 ubiquitin-protein ligase NEDD4-like protein R.S*LSSPTVTLSAPLEGAK.D(+2) XP_574161 0.3 ± 0.09 450 (Nedd4-2) (NEDD4.2) PREDICTED: E3 ubiquitin-protein ligase NEDD4-like protein R.SLS*SPTVTLSAPLEGAK.D(+2) XP_574161 0.3 ± 0.09 451 (Nedd4-2) (NEDD4.2) 452 hypothetical protein LOC300783 K.RPLFLAPDFDRWLDES*DAEMELR.A(+3) NP_001100301 0.31 ± 0.21 453 aquaporin 1 K.VWTSGQVEEYDLDADDINS*RVEMKPK.-(+4) NP_036910 0.31 ± 0.38 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 R.SNS*FSDEREFSAPSTPTGTLEFAGGEGK.G(+3) XP_001078032 0.31 ± 0.11 454 PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 R.SNSFS*DEREFSAPSTPTGTLEFAGGEGK.G(+3) XP_001078032 0.31 ± 0.11 455 catenin (cadherin associated protein), delta 1 R.FHPEPYGLEDDQRSMGY*DDLDYGMMSDYGTAR. NP_001101210 0.32 ± 0.14 456 R(+4) PREDICTED: AHNAK nucleoprotein isoform 1 isoform 1 R.S*NSFSDEREFSAPSTPTGTLEFAGGEGK.G(+3) XP_001078032 0.32 ± 0.11 457 PREDICTED: pleckstrin homology domain containing, family R.RPS*DRS*PTSVEKR.M(+3) XP_001080822 0.32 ± 0.27 458 G, member 3 PCTAIRE protein kinase 3 R.RLS*LSVPRPETIEESLTEFTEQFNQLHTQR.N(+4) NP_001093976 0.33 ± 0.33 459

460 hippocalcin K.QNS*KLRPEM#LQDLR.E(+2) NP_058818 0.33 ± 0.15 461 dyskeratosis congenita 1, dyskerin R.KRDS*DS*DADEATPTTTPR.V(+3) NP_596910 0.33 ± 0.24 PREDICTED: heterogeneous nuclear ribonucleoprotein U R.S*KSPPPPEEEAKDEEEDQTLVNLDTYTSDLHFQIS XP_001073417 0.34 ± 0.26 462 isoform a K.D(+4) complement receptor related protein isoform 1 K.EDSCVQPQSLLTSQENNSTSSPARNSLT*QEV.- NP_001005330 0.35 ± 0.17 463 (+3) PREDICTED: nuclear mitotic apparatus protein 1 R.AAQLQGS*PAPEKGEVLGDALQLDTLKQEAAK.L(+ XP_218972 0.35 ± 0.23 464 3) 465 myosin, heavy chain 9, non-muscle R.KGT*GDCS*DEEVDGKADGADAK.A(+3) NP_037326 0.37 ± 0.12 466 MYST histone acetyltransferase (monocytic leukemia) 3 K.QS*T*VS*K.G(+1) NP_001094040 0.38 ± 0.43 467 splicing factor, arginine/serine-rich 10 R.RRS*PS*PYYSR.G(+3) NP_476460 0.38 ± 0.25 468 phosphofructokinase, liver R.RTLS*IDKGF.-(+2) NP_037322 0.39 ± 0.18 469 phosphofructokinase, liver R.TLS*IDKGF.-(+2) NP_037322 0.39 ± 0.09 solute carrier family 14 (urea transporter), member 2 isoform K.LYESELSSPT*WPSSSQDTHPALPLLEMPEEKDLR. NP_808877 0.39 ± 0.25 470 2 S(+4) 471 aquaporin 1 K.VWTSGQVEEYDLDADDINS*RVEMKPK.-(+3) NP_036910 0.4 ± 0.31 tight junction protein 2 R.LYQDTRGS*YGS*DPEEEEEYRQQLAAHSK.R(+4) NP_446225 0.4 ± 0.16 472 ATP-binding cassette, sub-family C (CFTR/MRP), member 4 K.SGVDFGSLLKKENEEAEPS*PVPGT*PTLR.N(+3) NP_596902 0.4 ± 0.88 473

474 PREDICTED: topoisomerase (DNA) II beta K.KT*SFDQDS*DVDIFPSDFTSEPPALPR.T(+3) XP_001067768 0.4 ± 0.17 PREDICTED: heterogeneous nuclear ribonucleoprotein U R.SKS*PPPPEEEAKDEEEDQTLVNLDTYTSDLHFQIS XP_001073417 0.4 ± 0.28 475 isoform a K.D(+4) 476 PCTAIRE protein kinase 3 K.RLS*LPMDIR.L(+2) NP_001093976 0.41 ± 0.14 oxysterol binding protein K.MLAES*DES*GDEESVSQTDKTELQNTLR.T(+3) NP_001102397 0.44 ± 0.36 477

478 aquaporin 2 (collecting duct) R.RQSVELHS*PQS*LPR.G(+4) NP_037041 0.44 ± 0.18 nuclear casein kinase and cyclin-dependent kinase substrate K.NSQEDS*EDS*EEKDVK.T(+2) NP_073636 0.44 ± 0.45 479 1 nuclear casein kinase and cyclin-dependent kinase substrate K.NSQEDS*EDS*EEKDVK.T(+3) NP_073636 0.5 ± 0.52 480 1 catenin (cadherin associated protein), delta 1 R.FHPEPYGLEDDQRS*MGYDDLDYGMMSDYGTAR. NP_001101210 0.52 ± 0.16 481 R(+3) 482 aquaporin 2 (collecting duct) R.RQS*VELHS*PQSLPR.G(+3) NP_037041 0.55 ± 0.06 483 mitogen activated protein kinase 1 R.VADPDHDHTGFLT*EY*VATR.W(+3) NP_446294 0.58 ± 0.07 of dDAVP. with theSer-10residuephosphorylated. There isnochangeinabundancethepresence Supplementary Figure1:

Signal Intensity (x10,000) 10.4 41.6 10.4 41.6 + dDAVP - dDAVP Reconstructed ion chromatograms foraUT-A1/3 Reconstructedion phosphopeptide 29.80 Ser(p)-10-UT-A1/3 Time (min) 31.20 Ser(p)-62/63-UT-A1/3

194 - dDAVP

48.6

194 + dDAVP Signal Intensity (x10,000)

48.6

24.15 25.20 25.90 Time (min)

Supplementary Figure 2: Reconstructed ion chromatograms for a UT-A1/3 phosphopeptide with both Ser-62 and Ser-63 phosphorylated. There is no change in abundance in the presence of dDAVP.