Fig. 3

Effect of S81L mutation on protein expression of Gβ5 and RGS9-2 complex. a Cartoon representation of RGS9-Gβ5 complex crystal structure (PDB ID:2PBI) with S81L mutation shown in the red sphere. RGS9 and Gβ5 are shown in gray and cyan, respectively. b Cartoon representation of Gβ5 alone (PDB ID:2PBI) with S81 mutation shown in the red sphere. Residue S81 is present on β-strand S2β2 of WD1 repeat. WD1 and the neighboring WD2 repeats are represented in blue and orange, respectively. c The hydrogen bond formation of side chain of S81 (red) with backbone of V108 (orange) is shown as a dotted black line. The substituted residue L81 (green stick) will not be able to form a hydrogen bond; instead its bulkier side chain will have steric clashes with neighboring amino acids (V87, V108, C111, and C122 represented in stick). All structural representations are made using PyMOL software https://www.pymol.org. d Summary of three qRT-PCR experiments (each performed in triplicates) using patient and control LCL to determine the relative abundance of GNB5 in patient vs. controls showing no significant difference (two-tailed t-test p value 0.67). e, f Western blot analysis of GNB5 expression in patient lymphoblastoid cells compared to normal control. g Immunoblot analysis of protein expression in HEK293T/17 cells. RGS9-2, Gβ5, and R7BP were expressed in different combinations. The proteins extracted from the transfected cells used in BRET assay were subjected to immunoblot analysis using the indicated specific antibodies. Anti-GAPDH antibody was used as a loading control. Representative experiment out of three independent evaluations is shown