Figure 5

PI3Kα and γ isoforms associate with CNGA1 through their RA domain, examined using membrane-based yeast two-hybrid analyses. DSY-1 yeast cells co-expressing pDHB1-CNGA1 and pAlg5-NubG as negative control, CNGA1 + Grb14 as positive control, and CNGA1 + RA-PI3Kα and γ isoforms, respectively. The membrane and cytosolic fractions of yeast were subjected to immunoblot analysis with anti-CNGA1 and anti-G6PDH antibodies (A). All other expressed proteins (either alone or co-expressed) were immunoblotted with anti-CNGA1 antibody (B). Transformants were assayed for β-galactosidase assay by colony color by filter lift assay (C) or quantitative determination of β-galactosidase activity by the solution assay (D). Data are mean ± SD, n = 6, *p < 0.001 (significance between RA-PI3Kγ compared to RA-PI3Kα).