Figure 5.
HSC-dependent and Gαs-dependent signaling downstream of Smo are separable and distinct. All data represent mean ± S.D. t test: ***, p < 0.001; n.s., not significant. A, EPAC-BRET assay of cAMP levels in mock-transfected cells (−) and cells expressing SmoSD-GFP or a mutated form of SmoSD from which the C-terminal Fu-binding domain was deleted (SmoSDΔFu-GFP). B, immunoblot (IB) analysis of cells treated as in A. Blot was probed with anti-Smo antibody to reveal SmoSD-GFP variant expression and anti-α-tubulin as a loading control. C, ptc-luc reporter assay of cells as in A. D, EPAC-BRET assay of cAMP levels in control (−) or SmoSD-GFP expressing cells treated with β-gal (control) or fu dsRNA. E, immunoblot analysis of cells treated as in D. Blot was probed with anti-Smo antibody to reveal SmoSD-GFP expression and anti-α-tubulin as a loading control. F, ptc-luc reporter assay of cells as in D. G, ptc-luc reporter assay of mock- (−) or SmoSD-transfected cells, co-transfected with either empty vector (control) or mycCSBD. H, immunoblot analysis of cells treated as in G. Blot was probed with anti-GFP antibody to reveal SmoSD-GFP expression, with anti-myc tag to reveal mycCSBD, and anti-α-tubulin as a loading control. I, EPAC-BRET assay of cAMP levels in cells treated as in G. J, EPAC-BRET assay of cAMP levels in mock-transfected cells (−) and cells transfected with GαsQ215L, SmoSD-GFP, SmoSD.c14A-GFP, or SmoSD.c14A-GFP + GαsQ215L. K, ptc-luc reporter assay of cells as in J.