Supplementary Figure 6: Characterization of SNP rs11676272 (p.Ser107Pro; mutant V).

a, cAMP generation in BHK cells stimulated by forskolin (1 μM, in the presence of 100 μM IBMX). An asterisk indicates significant differences: CON (n = 18) vs. WT (n = 18) P < 0.001, CON (n = 18) vs. mutant V (n = 8) P < 0.001; no significant difference between WT and mutant V (P = 0.054). Means are represented as horizontal bars, s.d. as boxes, s.e.m. as whiskers and points represent individual data. b, Western blot analysis for ADCY3 and tubulin (TUB) from transiently transfected BHK cells with Myc-tagged hADCY3wt (WT) or plasmid encoding ADCY3 carrying the variant rs11676272 (V). Molecular weight standards are given in kDa. Expression levels of mutant V were 70 + 41.4% of WT levels (n = 7–12). c, CHO cells were transfected for 44 h with CreLuc reporter and either empty vector (CON) or plasmid encoding either Myc-tagged hADCY3wt (WT) or ADCY3 carrying the variant rs11676272 (V). Thereafter, culture medium was replaced and cells were cultured with either a medium containing 2% FCS (unstimulated) or a medium containing 2% FCS and 1 μM forskolin plus 100 μM IBMX (stimulated) for 4 h. Luciferase activity was measured and normalized to β-galactosidase activity. The data are expressed as the mean ± s.e.m. of four independent experiments performed in triplicate. An asterisk indicates significant differences: WT vs. CON P = 0.032, CON vs. V P = 0.003, WT vs. V P = 0.66.