Figure 3.

R7BP is required to detect interaction between Gα₁₃ and R7-RGS complexes containing any R7-RGS family member. A, proof of concept that split-luciferase complementation detects protein-protein interactions specific for Gα subunit identity and activity state. Data are represented as the -fold difference in normalized split-luciferase signal relative to the positive control for each Gα-CBGN construct. Data shown are the average of three independent experiments. B, split-luciferase complementation between the indicated Gα-CBGN proteins and CBGC-Gβ₅·RGS7 with or without R7BP. Each box represents a data point from one of three independent experiments. C, split-luciferase complementation between Gα₁₃(Q/L)-CBGN and CBGC-3xFLAG-Gβ₅ co-expressed with each R7-RGS isoform with or without R7BP. Luciferase signals were normalized to the level of CBGC-3xFLAG-Gβ₅ expression determined by immunoblotting. Each box represents a data point from one of five independent experiments. D, representative FLAG Western blot (WB) showing CBGC-3xFLAG-Gβ₅ and 3xFLAG-R7BP expression for each condition in C. The band marked with an asterisk is a degradation product of CBGC-3xFLAG-Gβ₅. E, Gα₁₃(Q/L)-CBGN-EE expression does not change with co-expression of R7-RGS heterotrimer proteins. Shown are representative immunoblots for C. Error bars indicate standard deviation of the mean. Statistical significance was determined using two-tailed, two-sample Student's t test assuming equal or unequal variance as deemed appropriate by F-test analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., p > 0.05.