Assembly competence of RIC8A mutants in vitro. (A and C) BN PAGE of RIC8A-knockdown complex (KD) reconstituted with indicated mutant proteins. WT, complexes from uninduced cells, Coomassie stain. (B) Western blot of gel run in parallel to (A), probed with anti-RIC8A antiserum (1:50). The amount of protein loaded on this gel was about one-third that in (A). RKD, knockdown RIC subcomplex; VKD, complex V from KD cells; RRC, reconstituted RIC. (D) SDS–PAGE profile of RKD and RRC (H1) (RIC reconstituted with H1 protein), compared to wild-type RIC (R). RIC subunits are numbered as in ref. 9.