Figure 4.

cAMP regulation is mediated by the evolutionarily conserved core of Smo. Graphed data represent mean ± S.D. t test: **, p < 0.01; ***, p < 0.001; n.s., not significant. A and B, ptc-luc reporter assay (A) and EPAC-BRET assay measuring cAMP levels (B) in mock-transfected cells (−), cells expressing full-length (Smo^SD-GFP), and cells expressing the C-terminally truncated (at amino acid 663) conserved core form of Smo (Smo^core-GFP) with or without Ptc co-expression. C, immunoblot (IB) analysis of cells treated as in A and B. Blot was probed with anti-GFP antibody to reveal Smo^SD-GFP or Smo^core-GFP expression and anti-α-tubulin as a loading control. D, EPAC-BRET assay of cAMP levels in mock (−) or Smo^core-GFP-transfected cells treated with dsRNA targeting β-gal (control) or Gαs. E, immunoblot analysis of cells treated as in D. Blot was probed with anti-GFP antibody to reveal Smo^core-GFP expression and anti-α-tubulin as a loading control. F, ptc-luc reporter assay in mock-transfected cells (−) and cells expressing Smo^core-GFP or the Gprk2 phosphosite mutant Smo^core.c1–3A-GFP. G, EPAC-BRET assay of mock transfected cells (−) and cells expressing Smo^core-GFP, treated with dsRNA targeting β-gal (control) or gprk2. H, immunoblot analysis of cells treated as in G. Blot was probed with anti-GFP antibody to reveal Smo^core-GFP expression and anti-α-tubulin as a loading control. I, EPAC-BRET assay measuring cAMP levels in mock-transfected cells (−) and cells expressing Smo^core-GFP or Smo^core.c1–3A-GFP. J, immunoblot analysis of cells treated as in I. Blot was probed with anti-GFP antibody to reveal Smo^core-GFP expression and anti-α-tubulin as a loading control. K, EPAC-BRET assay of cAMP levels in cells transfected with the indicated C-terminally truncated GFP-tagged Smo variants. L, immunoblot analysis of cells treated as in K. Blot was probed with anti-GFP antibody to reveal GFP-tagged Smo variants and anti-α-tubulin as a loading control.