Briefly, glass coverslips containing cells of interest were washed in PBS and fixed with either 4% PFA answer for 15 min at room heat or on snow, or with ice-cold methanol for 10C12 min at ?20C. to FLAG immunoprecipitation?(IP) and pellets analyzed by mass spectrometry. elife-63731-fig3-data1.xlsx (564K) GUID:?258EA8DC-E655-44FD-8B69-40ACCAF284AE Number 3source data 2: Initial western blots related to Figure 3B. Upper remaining, input Myc blot; top middle, input 100 kDa FLAG blot; top right, immunoprecipitation?(IP) Myc and 100 kDa FLAG blots; bottom left, input 35 kDa FLAG blot; bottom right, IP 35 kDa FLAG blot. elife-63731-fig3-data2.pdf (4.0M) GUID:?B6A8D433-3DEA-456D-9B6F-3640DB1F559E Number 3source data 3: Initial western blots related to Figure 3C. Upper remaining, SB269652 input Myc blot; top middle, immunoprecipitation?(IP) Myc blot; top right, input GFP blot; bottom remaining, IP GFP blot; bottom middle, input FOP blot; bottom right, IP FOP blot. elife-63731-fig3-data3.pdf (17M) GUID:?4AE4FE32-10FA-4DE0-A8C5-BA32AAD4F6A3 Figure 3source data 4: Initial western blots related to Figure 3D. Upper remaining, input Myc blot; top right, immunoprecipitation?(IP) Myc blot; lower remaining, input GFP blot; lower right, IP GFP blot. elife-63731-fig3-data4.pdf (29M) GUID:?983F7D2E-E99D-453F-BB03-BAC96DFDE100 Figure 3figure product 1source data 1: Original western blots for Figure 3figure product 1A. Top row SB269652 from remaining to right: input VPRBP blot, immunoprecipitation?(IP) VPRBP blot, input Myc Rabbit polyclonal to HHIPL2 blot. Second row from remaining to right: IP Myc blot, input FOP blot, IP FOP blot. Third row from remaining to right: input GFP blot (top), IP GFP blot (top). Fourth row remaining to right: input GFP blot (lower), IP GFP blot (lower). elife-63731-fig3-figsupp1-data1.pdf (20M) GUID:?50123CDA-E8FD-4FFD-9A44-67C056791F0F Number 3figure product 1source data 2: Initial western blots for Number 3figure product 1B. Top row, input VPRBP blot; second row, immunoprecipitation?(IP) VPRBP blot; third row remaining, input Myc blot; third row right, IP Myc blot; fourth row left, input GFP blots; fourth row right, IP GFP blots. elife-63731-fig3-figsupp1-data2.pdf (2.2M) GUID:?64E74894-4540-458C-937D-35FBC86FE6CE SB269652 Number 4source data 1: Initial western blots for Number 4B. Remaining, CEP78 blot; right, -tubulin blot. elife-63731-fig4-data1.pdf (4.1M) GUID:?7C372A73-2AFF-4D75-91F9-346AF2888DF6 Number 6source data 1: Initial western blots for Number 6A. Remaining, VPRBP blot; right, -tubulin blot. elife-63731-fig6-data1.pdf (3.9M) GUID:?5131B752-FC4E-43E8-B601-E38C1E4E0150 Figure 7source data 1: Initial western blots for Figure 7A. Remaining, CP110 blot; right, GAPDH blot. elife-63731-fig7-data1.pdf (4.4M) GUID:?CC166CE3-1C86-47AA-9FD6-7584C808BED0 Figure 7figure product 1source data 1: SB269652 Initial western blots for Figure 7figure product 1. Remaining, CP110 (top) and GAPDH blots in (A). Right, CP110 (top) and ?tubulin (lower) blots in (C). elife-63731-fig7-figsupp1-data1.pdf (4.4M) GUID:?5BD99BCC-C616-4D22-88DD-24723E1C12E2 Number 7figure product 2source data 1: Initial western blots for Number 7figure product 2A. Top, CP110 blot; bottom, -tubulin blot. elife-63731-fig7-figsupp2-data1.pdf (5.5M) GUID:?CF18D997-B664-483E-B461-51A0E1B8BAC1 Number 7figure supplement 3source data 1: Natural RNA-seq data for Number 7figure supplement 3. elife-63731-fig7-figsupp3-data1.xlsx (3.4M) GUID:?DA79A411-7A34-4985-B31E-BDD69E88CDEA Number 7figure product 4source data 1: Initial western blots for Number 7figure product 4A. Upper remaining, wildtype?(WT) CP110 blot; top right, WT GAPDH blot. Lower remaining, knockout?(KO) CP110 blot; lower right, KO GAPDH blot. elife-63731-fig7-figsupp4-data1.pdf (4.1M) GUID:?529A29D5-6751-4143-808D-4D13DD36B3F8 Figure 8source data 1: Original bots for Figure 8A. Remaining, CP110 blot; right, -tubulin blot. elife-63731-fig8-data1.pdf (10M) GUID:?162482C0-5C02-460C-91A8-13D63F0D93AE Transparent reporting form. elife-63731-transrepform.pdf (353K) GUID:?B992ACD6-DEF0-4D83-913F-EAC4BEC777AA Data Availability StatementAll data generated or analysed during this study are included in the manuscript and encouraging documents. Resource data files have been offered for Number 3A and Number 7-number product 2. Abstract CEP78 is definitely a centrosomal protein implicated in ciliogenesis and ciliary size control, and mutations in the gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is definitely unknown. Based on a recently found out disease-causing p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is definitely involved in CP110 ubiquitination and degradation, and recognized a novel connection between CEP78 and CEP350 that is weakened from the CEP78L150S mutation. We.