Fig. 3: The RGβ hug domain is required for RGα2 GAP activity.

a Co-immunoprecipitations of RGα2 and RGβ full-length (RGα2^fl, RGβ^fl) and hug domain deletion constructs (RGα2^Δhug, RGβ^Δhug) from transiently transfected HEK293T cells. b Co-immunoprecipitations of RGα2 with RGβ full-length and a construct only comprising the β-hug domain (RGβ^hug, aa 973-1118) from transiently transfected HEK293T cells. c Sec5 pull-down from HEK293T cells transfected with RalA and RGα2 without or with RGβ^fl and RGβ^Δhug. d Sec5 pull-down analysis of RGβ knock-out MEFs reconstituted with RGβ^fl or RGβ^Δhug. e In vitro GAP assay of RalA GTP hydrolysis by RGα2 without or with various RGβ truncation constructs. The same molarity of RGα2 was used in each assay and the activity was normalized to the amount of RGβ present in the individual reactions, n = 4 independent experiments, two-tailed t-test with Welch-correction, *p = 0.0358 (RGα2- RGα2/RGβ^hug); *p = 0.0140 (RGα2-RGα2/RGβ^SD-hug-pGAP); *p = 0.0146 (RGα2-RGα2/RGβ^{cHEAT-SD-hug-pGAP}:); *p = 0.0146 (RGα2-RGα2/RGβ^fl); data are represented as means ± standard deviation.