Anti rhoa antibody

Total protein samples were extracted from cells after incubating with valsartan, tetrandrine and Y-27632 for 48 h or with U73122 for 10 min. RIPA lysis buffer (Beyotime) with 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime) was used for cell lysis. The supernatant was transferred to a centrifuge tube for vortexing. Protein lysates were centrifuged at 4°C, 12,000 × g for 10 min. Protein samples were subjected to 10% SDS-PAGE for separation and then transferred onto polyvinylidene difluoride (PVDF) membranes according to standards methods. PVDF membranes were blocked with skim milk powder, and incubated with primary antibodies. against TRPC6 (1: 1000, Santa Cruz), Synaptopodin (1: 100; Santa Cruz), Nephrin (1: 100; Proteintech), ROCK1 (1: 1000; Proteintech), anti-RhoA antibody (1: 1000; Proteintech, Wuhan, China), and β-actin (1: 1000; Santa Cruz) at 4°C overnight. Activated RhoA was determined by measuring membrane-bound RhoA (GTP-RhoA) using the GST rhotekin-Rho binding domain that specifically pulls down activated RhoA, Secondary antibodies were peroxidase AffiniPure donkey anti-mouse or goat anti-rabbit IgG (H+L; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The blots were developed using ECL system (Share-bio, Shanghai, China) and analyzed using the TanonImage software (Tanon, Shanghai, China).