Equal amounts of protein were separated via SDS-PAGE using a 1.5-cm 4 to 12% bis-tris gel (Thermo Fisher Scientific, NP0336) or 1-cm 4 to 12% bis-tris gel for ProQ diamond stain with 1× Mops running buffer (Thermo Fisher Scientific, NP0001), transferred onto a polyvinylidene difluoride membrane (EMD Millipore, IPFL07810), blocked for 1 hour in 2% (v/v) BSA (Bioworld, 40220068-1) in TBST (tris-buffered saline with 0.3% Tween 20), and probed overnight with primary antibodies diluted in the above blocking buffer. Following extensive washes, HRP-linked enhanced chemiluminescence (ECL)–conjugated secondary antibodies, goat anti-rabbit IgG and rabbit anti-mouse IgG, were used to identify proteins of interest and were visualized with a SuperSignal West Pico PLUS ECL substrate (Thermo Fisher Scientific, 34579).
For in vitro binding assays with GST-Rho1, in vitro translated RhoGEF2 PH domains were detected on immunoblots with streptavidin-HRP (1:5000; Thermo Fisher Scientific, SA10001) and visualized with a SuperSignal West Pico PLUS ECL substrate (Thermo Fisher Scientific, 34579). The membranes were subsequently incubated with hydrogen peroxide to quench the bound HRP, washed, and subjected to GST immunoblotting using the above protocol.
For in vitro binding assays with GST-Rho1, in vitro translated RhoGEF2 PH domains were detected on immunoblots with streptavidin-HRP (1:5000; Thermo Fisher Scientific, SA10001) and visualized with a SuperSignal West Pico PLUS ECL substrate (Thermo Fisher Scientific, 34579). The membranes were subsequently incubated with hydrogen peroxide to quench the bound HRP, washed, and subjected to GST immunoblotting using the above protocol.