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Mukhopadhyay Supplemental Text Page 1 Inventory Of 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.
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