Journal: bioRxiv
Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling
doi: 10.1101/2025.01.06.631593
Figure Lengend Snippet: (A) Relative Git1 intensity in the Smo-TurboID proteomic results. (B) Git1 is biotinylated by Smo-TurboID independent of Shh treatment. Smo-V5-TurboID stable cells were infected with lentiviruses expressing YFP-Git1. Cells were treated with Shh for 1h, and cell lysates were used for purification with Streptavidin beads. a-Tubulin is used as loading control. (C) Git1 localizes to the basal body. NIH3T3 cells were transfected with YFP-Git1, and stained for primary cilium (Arl13b, red), basal body (Pericentrin, magenta), and nucleus (DAPI, blue). In addition to its putative distribution in the cytosol, Git1 also localizes to the basal body. Scale bar, 5 μm, 2 μm (inset). (D, F) Immunofluorescence imaging of Smo and phosphorylated Smo (pSmo) in WT and Git1-null cells. The cells are treated with 1 μg/ml recombinant Shh or vehicle. Primary cilium is highlighted by acetylated Tubulin (red). Scale bar, 5 μm, 2 μm (inset). (E, G) Quantification of ciliary Smo and pSmo immunofluorescence intensity (AU). n = 100-150 cells/condition from three biological replicates. (H) Representative images of immunofluorescence staining in Smo-TurboID cells transfected with control shRNA or shRNA against Git1. Cells were fixed 72hr after lentiviral infection and stained with PKA-C (green), Arl13b (red) and nucleus (DAPI, blue). Scale bar, 5 μm, 2 μm (inset). (I) Left: Quantification of ciliary PKA-C immunofluorescence intensity (AU), n = 90 cells/condition from three biological replicates; Right: Quantification of % ciliary PKA-C relative to total nucleus in the field. n= 15 fields per condition. Statistics in E, G, I (Left): two-way ANOVA followed by Tukey’s multiple comparison test. Statistics in I (Right) is assessed by one-way ANOVA followed by Sidak’s multiple comparison test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.
Article Snippet: The following antibodies were used in this study: Rabbit anti-pSmo (7TM antibodies, 7TM0239A-IC), Rabbit anti-Smo (gift from M. Scotts, Stanford University), Mouse anti-acetylated tubulin (Sigma,T6793), Rabbit anti-Arl13b (Proteintech, 17711–1-AP), Rat anti-Arl13b (BiCell Scientific, 90413), rabbit anti-IFT88 (Proteintech, 13967-1-AP), Goat anti-Gli2 (R&D Systems, AF3635,), Goat anti-Gli1 (R&D Systems, AF3455), Rabbit anti-Git1 (Novus Biologicals, NBP1-86144), Chicken anti-GFP (Aves labs, GFP-1020), Rabbit anti-GFP (Thermo Fisher Scientific, A-11122), Mouse-anti-Flag (Sigma, F3165), Rabbit anti-HA (Cell Signaling Technology, 3724), Mouse anti-GAPDH (Thermo Fisher Scientific, MA5-15738), Mouse anti-V5 (Thermo Fisher Scientific, R960-25), Mouse anti-PKACα (BD Biosciences, 610980), Rabbit anti-PKACα ( Cell Signaling, D38C6), Mouse-anti-pericentrin (BD Biosciences, 611814), Mouse anti-gamma Tubulin (Proteintech, 66320-1-Ig), DAPI ((Thermo Fisher Scientific, D21490), Donkey anti-rabbit Rhodamine (Jackson ImmunoResearch Labs, 711-025-152), Donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch Labs, 711-545-152), Donkey anti-mouse Alexa 488 (Jackson ImmunoResearch Labs, 715-025-151), Donkey anti-mouse Rhodamine (Jackson ImmunoResearch Labs, 715-545-151), Donkey anti-Chicken AlexaFluor 488 (Jackson ImmunoResearch Labs, 703-545-155), Donkey anti-Goat AlexaFluor 488 (Jackson ImmunoResearch Labs, 705-545-003), Donkey anti-Goat AlexaFluro 647 (Jackson ImmunoResearch Labs, 705-605-147), Alexa Fluor 647 Streptavidin (Jackson ImmunoResearch Labs, 016-600-084), Donkey-anti, HRP-Conjugated Streptavidin (Thermo Fisher Scientific, N100).
Techniques: Infection, Expressing, Purification, Control, Transfection, Staining, Immunofluorescence, Imaging, Recombinant, shRNA, Comparison
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