FIGURE 5.

Gpr175 functions at the level of PKA. A, Gli-luciferase (Gli-Luc) activity in S12 cells depleted (with siRNA pools) of SuFu or Gpr175 alone or in combination in the absence of Hh (expressed as a percentage of siSuFu). Mean ± S.D. of three independent experiments are plotted (*, p ≤ 0.05). B, representative Western blot (of n = 3) with goat anti-SUFU, rabbit anti-Gpr175, and anti-tubulin (loading control) antibodies following SuFu and/or Gpr175 knockdown in S12 cell lysates (40 μg/lane). Note that knockdown of each protein (100% for Gpr175 and 57 ± 5.7% for SuFu; n = 3) was unaffected by the presence of the second siRNA. C, as in A but with siGli3 and siIft88 (pools of 4 siRNAs) as a positive control. Error bars represent S.D. of the mean of five independent experiments (**, p ≤ 0.01). D, a representative of 3 Western blots with mouse anti-Gli3, rabbit anti-Gpr175, and anti-tubulin (loading control) antibodies showing endogenous expression of Gli3 (full-length (73 ± 23% depleted) and repressor form (96 ± 2.8% depleted)) and Gpr175 (100% depleted) in S12 cell lysates (40 μg/lane) that were treated with siGli3 or siGpr175 alone or in combination. LE, longer exposure. E, S12 cells were stimulated with 80 μm PKA inhibitor 14–22 amide (or DMSO vehicle control) for 24 h in the absence of Hh following siGpr175 (gray) or non-targeting control (white) transfection (n.s., not significant). F, as in E except Hh-stimulated S12 cells were inhibited with 200 nm Fsk or DMSO control for the last 24 h. Mean ± S.D. of 3 independent experiments normalized to siNTC + DMSO (*, p ≤ 0.05).