Mukhopadhyay Supplemental Text Page 1
Inventory of Supplementary information:
-Supplementary experimental procedures
-Supplementary figure legends and references
-Six supplementary figures
Mukhopadhyay Supplemental Text Page 2
Supplemental materials and methods
Antibodies and Reagents
Commercial antibodies used were against DYNC1H1 (sc-9115, Santa Cruz
Biotechnology), IQGAP1 (610612, BD Transduction Laboratories), DYNC1I2 (sc-
70977, Santa Cruz Biotechnolog), KIF3A (Sigma, K3513), IFT88 (13967-1-AP,
ProteinTech), α-tubulin (clone DM1A, T6199, Sigma), Glu-tubulin (AB3201, Millipore), acetylated α-tubulin (mAb 6-11B-1), γ-tubulin (GTU-88, ab11316, Abcam), rabbit pericentrin (ab4448, Abcam), Sstr3 (sc-11617, Santa Cruz Biotechnology), ACIII (sc-
588, Santa Cruz Biotechnology), β-tubulinIII (T8660, Sigma), c-Myc (clone 9E10; sc-
789, Santa Cruz Biotechnology) and GFP (ab290, Abcam; sc-9996, Santa Cruz
Biotechnology). Antibodies against recombinant GST were raised in rabbits and affinity purified (kind gift of Jagath Reddy Junutula). Fluorescent secondary antibodies for immunofluorescence were from Jackson ImmunoResearch or Invitrogen, while IRDye
700CW and IRDye 800CW secondary antibodies for immunoblotting were from Li-cor
Biosciences. Leptomycin B and U73122 were from Sigma.
Generation of stable cell lines
Stable clonal RPE or NIH 3T3 cell lines expressing LAPTUB isoform a, LAPTULP1,
LAPTULP2, LAPTULP3, LAPTULP3 (K268A, R270A), LAPTULP3 (R300A), MCHR1GFP and SSTR3GFP were generated by retroviral infection and clonal selection using FACS.
Polyclonal RPE and RPE LAPTULP3 cell lines stably expressing LAPTULP3 deletions or
MycTULP3 (1-183) (with or without mut12 mutations) respectively were generated by retroviral infection. Stable clonal Flp-in RPE cell lines expressing LAPTULP3 mut1,
LAPTULP3 mut12, or Flp-in IMCD-3 lines expressing LAPTULP3 were generated using
Mukhopadhyay Supplemental Text Page 3 protocols for integrating FRT containing constructs at a single locus (Invitrogen).
Plasmids and Proteins
Full-length cDNAs in the pENTR vector for TULP1, TULP2, and TULP3 were Ultimate
ORF clones from Invitrogen. ORF clones for MCHR1 (RG200997), SSTR3 (RG215219) were from Origene. IFT140, TUB-isoform b, TULP3 deletions, and TULP4 constructs were synthesized by DNA 2.0 in the Gateway vector pDONR221. TUB-isoform a was amplified from MGC IRAT clone 30915418 and subcloned into pDONR221
(Invitrogen). Expression constructs were generated by Gateway cloning into pG-LAP1
(pCMV-EGFP-TEV-Speptide-X with FRT) (Torres et al. 2009), pG-LAP5 (pEFα-X-
Speptide-PrecissionS-EGFP with FRT), pDEST GEX-6P1, pDEST MBP, pDEST chicken βactin promoter-EGFP-X or pDEST CS2-6xMyc. For retroviral infections, N- and C-terminal LAP-tagged, and C-terminal GFP-tagged clones were subcloned into pBABE Puro, while Myc-tagged TULP3 (1-183) and Myc-tagged TULP3 (1-183) mut12 were subcloned into pQCXIN (Clontech). Single or multiple amino acid mutations in full-length TULP3 and TULP3 deletions were generated using Quikchange site-directed mutagenesis kit (Stratagene). All constructs were verified by sequencing. MBPTULP3,
MBPTULP3mut12, GSTTULP3, HisTULP3 (1-183), and HisTULP3 (1-183) mut12 were expressed and purified using standard methods. siRNA Oligo Transfections, Reverse transcription and Quantitative PCR
S12 cells were seeded into 8-well LabTekII microscope slides and reverse-transfected with 100nM siRNA (for single knockdowns), or 75nM siRNA each (for double knockdowns) with DharmaFECT-2 (Dharmacon). After 48h, cells were shifted to 0.5%
Mukhopadhyay Supplemental Text Page 4 serum ± 200ng/ml octyl-Shh “Hh” (Taylor et al. 2001) for indicated periods. For qRT-
PCR, total RNA was prepared with the RNeasy plus Mini kit (Qiagen). RNA was used for qRT-PCR by using TaqMan one-step RT-PCR master mix reagents (Applied
Biosystems). TaqMan probes for qRT-PCR spanned corresponding exon segments and are inventoried in the manufacturer’s website (Applied Biosystems). Duplicate or triplicate reactions were run and analyzed on an ABI 7500 thermocycler using GAPDH or murine Rpl19 as the endogenous control.
Immunofluorescence and Microscopy
Qualitative assessment of the strength of Gli3 staining at cilia tips was performed as described previously (Wen et al. 2010).
Biochemical Fractionations
To fractionate total RPE cell extracts, cells were lysed in LAP150 buffer (50 mM HEPES
[pH 7.4], 150 mM KCl, 1 mM EGTA, 1 mM MgCl2, protease inhibitors) containing 0.3%
NP-40, followed by centrifugation at 100,000 × g for 1 hr. The resulting extract was fractionated on a Superose-6 10/300 GL column (GE Healthcare), and 0.5 ml fractions were concentrated by methanol/chloroform precipitation before immunoblotting.
In vitro protein binding assays
High spin lysates from TULP3 siRNA-treated IFT140LAP RPE cell pellets (~200 µl packed cell volume) were immunoprecipitated using anti-GFP antibody crosslinked to
Affi-Prep Protein A beads (Bio-Rad). The washed beads were treated overnight with
PrecissionS and the digests were eluted in a total of ~300 ul of LAP150N buffer (50 mM
HEPES [pH 7.4], 150 mM KCl, 1 mM EGTA, 1 mM MgCl2, 10% glycerol, 0.05%
NP40). Freshly prepared 60 µl of the PrecissionS eluates were added to 25 µl of
Mukhopadhyay Supplemental Text Page 5
MBPTULP3 or MBPTULP3 mut12 beads (40 µg of recombinant MBPTULP3 or MBPTULP3 mut12 immobilized on ~250 µl of packed Amylose resin) in the presence of recombinant
HisTULP3 (1-183) or HisTULP3 (1-183) mut12 [0.5 µg to 10µg] in 140 µl of LAP100 buffer (50 mM HEPES [pH 7.4], 100 mM KCl, 1 mM EGTA, 1 mM MgCl2, protease inhibitors, 10% glycerol) and incubated for 2 hours by mixing at 4°C. After 5 washes in
LAP100N (LAP100 with 0.05% NP40) buffer, MBPTULP3 or MBPTULP3 mut12 bound proteins were eluted in 45 µl of sample buffer.
Mass spectrometry
For mass spectrometry, lanes containing protein samples were each excised into 8-12 equal gel sections, reduced, carboxyamidomethylated and digested with trypsin. Peptide identification of each digestion mixture was performed by microcapillary reversed-phase high-performance liquid chromatography coupled with nanoelectrospray tandem mass spectrometry (µLC-MS/MS) on an LTQ-Orbitrap Velos mass spectrometer
(ThermoFisher Scientific). The Orbitrap repetitively surveyed an m/z range from 395 to
1600, while data-dependent MS/MS spectra on the twenty most abundant ions in each survey scan were acquired in the linear ion trap. MS/MS spectra were acquired with relative collision energy of 30%, 2.5-Da isolation width, and recurring ions dynamically excluded for 60 s. Preliminary sequencing of peptides was facilitated with the SEQUEST algorithm with a 30 ppm mass tolerance against a species specific (mouse or human) subset of the UniProt Knowledgebase. With a custom version of Proteomics Browser
Suite (ThermoFisher Scientific), peptide sequence matches (PSMs) were accepted with mass error <2.5ppm and score thresholds to attain an estimated false discovery rate of
~1% using a reverse decoy database strategy. Spectral counts were used to provide a
Mukhopadhyay Supplemental Text Page 6 preliminary comparison of proteins across different states/preparations.
PIP and membrane lipid blots
Bnding of recombinant proteins to pre-spotted PIP and membrane lipid strips (Echelon) was performed according to Dowler et al. 2002. After blocking strips in Licor blocking buffer for 1 hr at room temperature, the strips were incubated in Licor blocking buffer containing 1 µg/ml of recombinant GSTTULP3 or GST protein overnight at 4°C. After washing for three times in TBS-T, the blots were immunoblotted for GST.
Statistical Analyses
Statistical analyses of ciliation assays and receptor localization experiments were performed using one-way ANOVA and Tukey-Kramer’s post hoc multiple comparison tests between all possible pairs in each data set (Supplemental Fig. S1C), or between all possible pairs of Hg treated or untreated cells (Supplemental Fig. S5C,D) using JMP 8 statistical analyses package.
Mukhopadhyay Supplemental Text Page 7
Supplemental references
Cole, D.G. and Snell, W.J. 2009. SnapShot: Intraflagellar transport. Cell 137(4): 784-784 e781. Dowler, S., Kular, G., and Alessi, D.R. 2002. Protein lipid overlay assay. Sci STKE 2002(129): pl6. Efimenko, E., Blacque, O.E., Ou, G., Haycraft, C.J., Yoder, B.K., Scholey, J.M., Leroux, M.R., and Swoboda, P. 2006. Caenorhabditis elegans DYF-2, an orthologue of human WDR19, is a component of the intraflagellar transport machinery in sensory cilia. Mol Biol Cell 17(11): 4801-4811. Lin, B., White, J.T., Utleg, A.G., Wang, S., Ferguson, C., True, L.D., Vessella, R., Hood, L., and Nelson, P.S. 2003. Isolation and characterization of human and mouse WDR19,a novel WD-repeat protein exhibiting androgen-regulated expression in prostate epithelium. Genomics 82(3): 331-342. Taylor, F.R., Wen, D., Garber, E.A., Carmillo, A.N., Baker, D.P., Arduini, R.M., Williams, K.P., Weinreb, P.H., Rayhorn, P., Hronowski, X. et al. 2001. Enhanced potency of human Sonic hedgehog by hydrophobic modification. Biochemistry 40(14): 4359-4371. Torres, J.Z., Miller, J.J., and Jackson, P.K. 2009. High-throughput generation of tagged stable cell lines for proteomic analysis. Proteomics 9(10): 2888-2891. Tran, P.V., Haycraft, C.J., Besschetnova, T.Y., Turbe-Doan, A., Stottmann, R.W., Herron, B.J., Chesebro, A.L., Qiu, H., Scherz, P.J., Shah, J.V. et al. 2008. THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia. Nat Genet 40(4): 403-410. Wen, X., Lai, C.K., Evangelista, M., Hongo, J.A., de Sauvage, F.J., and Scales, S.J. 2010. Kinetics of hedgehog-dependent full-length Gli3 accumulation in primary cilia and subsequent degradation. Mol Cell Biol 30(8): 1910-1922.
Mukhopadhyay Supplemental Text Page 8
Supplemental figure legends
Supplemental Figure S1. TULP3 interacts with the IFT-A complex (A) Tandem affinity purification from clonal LAPTULP3 NIH 3T3 cells, treated ± Hh for 28 hours (Taylor et al., 2001). Eluates were resolved on a 4-12% Bis-Tris NuPAGE gel, and silver stained. Stagfusion proteins in each lane are marked by arrowheads. The IFT-A complex proteins are marked based on LC MS/MS analysis of gel slices.
(B) Table showing the peptide spectral count and % coverage of selected proteins identified by mass spectrometry in tandem purification eluates from clonal mouse NIH 3T3 cells expressing LAP-tagged human TULP3 treated ±Hh for 28 hours. The % coverage of LAPTULP3 is less than in Fig. 1 as it was compared to the mouse database of proteins. The corresponding Chlamydomonas (Cr), C. elegans (Ce), Drosophila (Dm), and human (Hs) names are mentioned to the left of the mouse (Ms) names (Cole and Snell 2009). As the Chlamydomonas names do not exactly correspond to the respective molecular weights, we mainly follow the human/mouse names in the text. THM1 and WDR19 are used instead of TTC21B and IFT144 respectively, as these names have been previously used in the literature (Tran et al. 2008; Lin et al. 2003; Efimenko et al. 2006). (C) TULP3 and IFT-A do not regulate cilia formation. RPE cells were transfected with indicated siRNAs for 72 hours, and serum starved for the last 24 hours before fixing and staining for pericentrin, Ac-tubulin, and DNA. Cilia were counted in 3-8 independent experiments. KIF3a knockdown was included as positive control. Error bars represent SEM. *, P<0.0001 with respect to control. Supplemental Figure S2. TULP3 associates with the “core” IFT-A complex (A) TULP3-associated proteins were purified from LAPTULP3 murine IMCD-3 cells as in Figure 1A, except following 72h depletion of the indicated subunits by siRNA. Silver stained gel (top panel) and western blots (lower panels) of tandem affinity purifications are shown. The IFT-A complex proteins are marked as in Figure 1A. (B) Efficiency of siRNA-mediated knockdown in RPE and IMCD-3 cells. Cells were treated with indicated OTP siRNAs for 72 hours, and lysates were processed for immunoblotting with indicated antibodies. The % of remaining proteins is shown below. For human TULP3, siRNAs 1 and 8 were not used further as they were toxic to the cells, while siRNAs 2, 3, and 7 resulted in almost complete depletion (ND). Murine Tulp3 siRNAs 1 and 2 caused almost complete depletion (ND). Note that human TULP3 (49.7 kDa) (marked with an arrow) is of slightly lower molecular weight than mouse Tulp3 (51.2 kDa) (marked with an arrowhead). For THM1, OTP2 and 3 resulted in maximal knockdown; however, OTP2 was toxic, and not used further.
Mukhopadhyay Supplemental Text Page 9
(C) RPE cells were transfected with indicated siRNAs for 72 hours and the relative levels of mRNA (as % of non-targeting control ± SD) were estimated using RT- qPCR. (D) LAPTULP3 localizes to cilia in IMCD-3 (upper panel) and NIH 3T3 cells (lower panel). LAPTULP3 clonal lines were serum starved for 24 hours before fixing and staining for Ac-tubulin (AcTub, magenta) and DNA (blue). Scale bar, 5 µm. (E) Knockdown of TULP3 does not affect IFT140 localization. RPE IFT140LAP clonal cells were depleted of TULP3 for 72h (bottom) or not (top), serum starved for the last 24 hours, before fixing and staining for pericentrin (PCNT, magenta), Ac-tubulin (AcTub, magenta), and DNA (blue). Scale bar, 5 µm. Supplemental Figure S3. TULP3 is a nucleo-cytoplasmic shuttling protein, and does not localize to cilia in IFT-A binding mutants (A) RPE LAPTULP3 line A cells were serum starved for 24 hours and treated with leptomycin B (10ng/ml, lower panels) or vehicle (0.2% methanol, upper panels) before fixing and staining for pericentrin (PCNT, red), Ac-tubulin (AcTub, magenta) and DNA (blue). White arrows mark cilia. Scale bars, 5 µm. (B) RPE LAPTULP3 K268A,R270A (LAPTULP3KR) cells were serum starved for 24 hours before fixing and staining for Ac-tubulin (AcTub, magenta) and DNA (blue). White arrows mark cilia. Scale bars, 5 µm. (C) RPE LAPTULP3 mut1, and LAPTULP3 mut12 clonal cells were serum starved for 24 hours before fixing and staining for pericentrin (PCNT, magenta), Ac-tubulin (AcTub, magenta) and DNA (blue). Scale bars, 5 µm. Supplemental Figure S4. Phylogenetic tree of Tubby family proteins Phylogenetic tree of selected Tubby family proteins from Treefam (www.treefam.org). Bootstrapping supports rescaled around 100 are indicated by hatchmarks: (/) – <50%; (//) – 51-80%; (///) – 81-100%.
Supplemental Figure S5. TULP3 or IFT-A regulate localization of a subset of ciliary GPCRs (A) RPE GFPRAB8a stable cells were transfected with indicated siRNAs for 78 hours, and serum starved for the last 30 hours before fixing and staining for pericentrin, Ac-tubulin, and DNA. Percentages of total cilia and GFP-positive cilia were counted. (B) Smo translocation assays on double knockdown of Tulp3 or IFT-A with Dync2h1. S12 cells were transfected with indicated siRNAs for 72 hours, and serum starved for last 18 hours ± Hh before fixing and staining for Smo, Ac-tubulin, and DNA. Error bars represent SD. *, P<0.05; blue and red asterisks denote significance with respect to control untreated or control Hg treated cells respectively.
Mukhopadhyay Supplemental Text Page 10
(C) Knockdown of Tulp3 and IFT-A complex do not affect Gli3 accumulation to the tips of the cilia on Hh stimulation. S12 cells were transfected with indicated siRNAs for 72 hours, serum starved for last 18 hours and stimulated ± Hh during the last 45 min before fixing and staining for Gli3, Ac-tubulin, and DNA. Gli3- positive cilia were scored as described in Methods. Error bars represent SD. (D) Gli3 processing assays in single or double knockdowns of Tulp3 with Dync2h1. S12 cells were transfected with indicated siRNAs for 72 hours, and serum starved ± Hh for the last 18 hours before lysing and immunoblotting for Gli3 and tubulin. The relative levels of mRNA (as % of non-targeting control) are shown in the table below. (E) Gli3 processing assays as in (D) with single or double knockdowns of Ift140 with Dync2h1. (F) Gli3 processing assays as in (D) with single or double knockdowns of Wdr19 with Dync2h1. Supplemental Figure S6. Both phosphoinositide and IFT-A binding properties of TULP3 regulate GPCR trafficking. (A) Binding of TULP3 to an intact preformed IFT-A complex in vitro. PrecissionS eluates from TULP3 siRNA-treated IFT140LAP RPE cells were added to MBPTULP3 or MBPTULP3 beads. MBPTULP3 or MBPTULP3 mut12 bound proteins and corresponding flowthroughs were silver-stained (upper panel) or immunoblotted for THM1 (lower panel). The anti-THM1 antibody cross-reacts with Stagfusion proteins, and allowed us to detect the IFT140Stag protein. The IFT- A complex proteins are marked as in Figure 1A. The MBPTULP3 or MBPTULP3 mut12 proteins are marked by arrowheads. (B) RPE LAPTULP3 cells stably expressing indicated myc-tagged N-terminal TULP3 proteins were serum starved for 30 hours before fixing, and immunostaining for myc, pericentrin, Glu-tubulin, and DNA. Percentages of GFP-positive cilia in myc-positive cells were counted. A chi-square test was used to test the statistical significance between the two distributions. *, P <0.0001. (C) Binding of GSTTULP3 (upper panels) and GST (lower panels) to PIP and membrane lipid strips. The abbreviations are as follows: BLK, Blank; Sulfatide, 3-sulfogalactosylceramide; PI, Phosphatidylinositol; PG, Phosphatidylglycerol; PC, Phosphatidylcholine; PE, Phosphatidylethanolamine; PS, Phosphatidylserine; PA, Phosphatidic Acid; DAG, Diacylglycerol; TG, Triglyceride; S1P, Sphingosine-1-Phosphate; LPC, Lysophosphocholine; LPA, Lysophosphatidic Acid.
150425_Mukhopadhyay_Figure S1
A TULP3 TULP3 LAP LAP ( - Hh) ( + Hh)
200kD Ift140 Wdr19, Thm1 Wdr35, Ift122 116kD 97kD
66kD
Stag 55kD TULP3
B
LAPTULP3 (-Hh) LAPTULP3 (+Hh) Total Total Peptides Peptides Protein Protein Protein (Unique (Unique (Cr) (Ce) Protein (Dm) (Hs) Protein (Ms) Gene ID MW (KDa) Peptides) % coverage Peptides) % coverage
--- TULP3 Tulp3 7289 49.7 105 (13) 17 84 (10) 15.2
IFT140 CHE-11 OSEG3/REMPA IFT140 Ift140 106633 165.9 127 (46) 31.3 74 (36) 26 IFT144 DYF-2 OSEG6 WDR19 Wdr19 213081 151.5 116 (49) 34.5 69 (34) 28.9 IFT139 ZK328.7 - THM1 Thm1/Ttc21b 73668 150.8 139 (45) 31.9 86 (39) 31.5 IFT121 IFTA-1 OSEG4 WDR35 Wdr35 74682 134, 132.8 96 (45) 34.5 79 (41) 36.9 IFT122 DAF-10 OSEG1 IFT122 Ift122/Wdr10 81896 134.9, 134.8 82 (34) 28.5 88 (31) 29.9 IFT43 - - IFT43 Ift43 76411 22.1 10 (6) 28.6 - -
C 70 60 50 40 30 20 *
Ciliated cells (%) 10 0 OTP2 OTP3 OTP7 THM1 KIF3A IFT140 IFT122 Control TULP3 WDR19 WDR35 siRNA treatment 150425_Mukhopadhyay_Figure S2
A siRNA
Wdr19
Thm1 Ift140 Wdr35 Ift122 Control 200kD
116kD 97kD
66kD
55kD
WB: Thm1
TULP3 51kD
C B RPE cells IMCD-3 cells RPE cells
siRNA Relative mRNA levels WB: TULP3 ► WB: THM1 Control 100 100 100 100 51kD WDR19 23.1±1.3 - - - WB: α-tubulin WB: α-tubulin IFT140 - 13.6±0.5 -- IFT122 - - 12.9±0.3 51kD 51kD WDR35 - -- 20±2 control control control siRNA 1 siRNA 2 siRNA 3 siRNA siRNA 4 siRNA 5 siRNA 6 siRNA 8 siRNA siRNA 7 siRNA 1 siRNA 2 siRNA 3 siRNA siRNA 1 siRNA 2 siRNA 3 siRNA siRNA 4 siRNA siRNA 4 siRNA % TULP3 100 ND ND ND 48 46 20 ND ND 100 ND ND 31 25 100 79 30 20 52 % THM1 remaining remaining
D E LAPTULP3 AcTub Merge / DNA IFT140LAP PCNT / AcTub Merge / DNA
IMCD-3 CONTROL
3T3 TULP3 si RNA 150425_Mukhopadhyay_Figure S3
A DNA LAPTULP3 PCNT AcTub Merge
0.2% Methanol
Leptomycin
B Ac DNA LAPTULP3KR Tub Merge
C GFP AcTub / PCNT Merge / DNA
LAPTULP3 mut1
LAPTULP3 mut12 150425_Mukhopadhyay_Figure S4
Os03g0351400 (Oryza) TULP1 (Human) TUB (Human)
TULP3 (Human)
TULP2 (Human) KINGTUBBY (Drosophila)
TUB-1 (C. elegans)
TULP4 (Human)
WDR35 (Human) Ciliated cells (%) GFP 200KDa 100KDa 150KDa levels mRNA Relative RAB8a + cilia (%) siRNA 75KDa 10 20 30 40 50 60 70 80 90 E C 0 Shh % Gli3 positive cilia A 10 20 30 40 50 60 70 80 90
0 N=148 Control 0 2 38 92 40 100 102 100 oto Dync2h1 Control - - - Control + + + + 0h 45minShh 0hr
TULP3 N=113
- - Tulp3 OTP3
N=140 Wdr19 OTP7 41 213 12 15 14 - - Ift140 Ift140 N=88 WDR19 Ift122 Ift140 Dync2h1, 132 31
- N=123 IFT140 Thm1 IFT122 N=111 Tubulin Gli3R Gli3FL Dync2h1 Ift140 Wdr35 N=107 THM1
WDR35 N=112 D 200KDa 100KDa 150KDa B 200KDa 100KDa 150KDa siRNA levels mRNA Relative 75KDa siRNA levels mRNA Relative 75KDa % Smo positive cilia 100 F 70 Shh 10 20 30 40 50 60 80 90 Shh 0
Control 150425_Mukhopadhyay_Figure S5 0 7 35 30 87 100 97 100 oto Dync2h1 Control oto Dync2h1 Control - - - 0 1 27 33 71 100 83 100 - - - Tulp3 + + + + + + + + Wdr19 Ift140 - - - -
Wdr35 16hrShh 0hr Wdr19, Ift140 41 614 16 12 14 - -
21 941 39 12 12 Wdr19, Ift122 Tulp3 - - Wdr19 Ift140, Wdr35 Ift140, Thm1 Tulp3 Dync2h1, 230 32 Wdr19 Dync2h1, Ift140, Tulp3 834 28 - - Ift122, Tulp3 * Dync2h1 * * Tubulin Gli3R Gli3FL Dync2h1 Tulp3 * Gli3R Gli3FL
Tubulin Dync2h1, Tulp3 Dync2h1 Wdr19 * Dync2h1, Wdr19 * * Dync2h1, Ift140 * Mukhopadhyay_Figure S6
A Stag
+ + + + + + IFT140Stageluate - + - - + - MBPTULP3 MBP 30% IFT140 eluate input - - + - - + TULP3 mut12
116kD
97kD ► ►
IFT140Stag THM1 97kD
Amylose bead Flowthrough Pulldown
B 120
100
80
60
40
20 TULP3 + cilia (%) *
LAP 0 N=28 N=50 MycTULP3 (1-183) MycTULP3 (1-183) sdm12
C
Sphingomyelin PI(3,4,5)P3PI(4,5)P2PI(4)P PI BLK Sulfatide Cholesterol BLK PS PA PI(3,4,5)P3PI(4,5)P2PI(3,5)P2PI(3,4)P2S1P TULP3 GST
PC PE PI PG PC PE PS PA TG LPC LPA DAG PI(5)P PI(4)P PI(3)P Cardiolipin
BLK Sulfatide SphingomyelinCholesterolPI(3,4,5)P3PI(4,5)P2PI(4)P PI BLK PS PA PI(3,4,5)P3PI(4,5)P2PI(3,5)P2PI(3,4)P2S1P GST GST
PC PI PG PC PE PS PA TG PE LPA DAG LPC PI(5)P PI(4)P PI(3)P Cardiolipin