FIGURE 7.
Gpr175 knockdown modestly increases levels of Gli3 repressor. A–C, representative Western blot of endogenous Gli3 showing Gli3-FL and Gli3-R bands in S12 cells transfected for 72 h with NTC, siIft88 (positive control), or siGpr175 with and without 24 h Hh treatment. Graphs show quantitations of Gli3-R (B) and Gli3-FL (C) normalized to NTC-Hh and tubulin (1A2) loading controls from three or more independent Western blots (*, p ≤ 0.05; **, p ≤ 0.01, n.s. not significant versus respective NTC). D, as in A but with siGαi1, also quantitated in B and C. E, representative images of control and Gpr175-depleted S12 cells showing localization of Gli3 (red) at the cilia tips (distal end of acetylated tubulin signal, green) from serum-starved cells without (left panel) or with 60 min Hh treatment (right panel) and counterstained with DAPI (blue). Scale bar is 1 μm. F, quantitation of the data in E as a percentage of cilia with Gli3 accumulated at the tips (mean ± S.D. of three independent experiments of ≥100 cilia each).