Figure 1. Co-dependence of heterotrimeric Golf subunits and AC5 in complex formation and expression.
(A) Immunoprecipitation of AC5 complexes from striatal lysates of wild type mice. Specific anti-AC5 antibodies but not non-immune IgGs pull down Gαolfβ2γ7 subunits from native striatal tissues. (B) Significant reduction of Gαolf expression in striatum tissue from mice lacking AC5. Total striatal lysates were analyzed by immunoblotting using indicated antibodies and quantified by densitometry. ***p<0.001, Student’s t-test, n = 3 mice. (C) Immunoprecipitation of AC5 from wild type (+/+) and Adcy5-/- tissues confirms specificity of AC5-Golf binding. Same anti-AC5 antibodies were used in both immunoprecipitaiton experiments and samples were processed in parallel. (D) Significant reduction of AC5 and Gαolf expression in striatal tissues from mice lacking Gγ7. Total striatal lysates from wild type and Gng7-/- mice were analyzed by immunoblotting with indicated antibodies and quantified by densitometry. *p<0.05, **p<0.01, Student’s t-test, n = 3 mice. (E) Significant reduction of AC5 and Gβ2 expression in striatal tissues from mice with reduced expression of Gαolf. Total striatal lysates from wild type and Gnal+/- mice were analyzed by immunoblotting with indicated antibodies and quantified by densitometry. *p<0.05, **p<0.01, ***p<0.001, Student’s t-test, n = 3 mice. (F) Mutual stabilization of AC5 and Gαolf upon co-transfection in HEK293 cells. Equal amounts of cDNAs were transfected into cells as described in the Methods section and protein expression was assessed by immunoblotting with specific antibodies (G) Lack of co-stabilization between AC5 and Gβ2γ7 subunits in co-transfected HEK293 cells. Indicated constructs were transfected into cells and changes in protein expression were monitored by immunoblotting with specific antibodies. (H) Mutual stabilization of Gαolf subunits with Gβ2γ7 subunits in co-transfected HEK293 cells. The experiment was conducted as described for panel G.