Gradient precast sds polyacrylamide gel electrophoresis

All lysis buffers were supplemented with 1% protease inhibitor (Sigma Aldrich, USA). Protein extracts were resolved using gradient precast SDS – polyacrylamide gel electrophoresis (Biorad Laboratories Inc, USA), and then electro-transferred onto a nitrocellulose membrane by using Trans-Blot® Turbo™ Transfer System (Biorad Laboratories Inc, USA) for immunoblot analysis. Antibody probing was done as per manufacturers’ instructions Antibodies used include anti-RhoA antibody (1:1000, Abcam, UK), anti-CDC42 antibody (1:1000, Abcam, UK), anti-Rac1 antibody (1:1000 Abcam, UK), anti-phospho-MYPT1 (Thr696) antibody (1:1000, Cell Signaling Technology, USA), anti-myosin light chain (phospho S20) antibody (1:1000, Abcam, UK), anti-GAPDH antibody (1:2000, Abcam, UK). Secondary antibodies either IRDye® 800CW Goat anti-Mouse IgG (LI-COR, USA) or IRDye® 800CW Goat anti-Rabbit IgG (LI-COR, USA) were used with dilution of 1:15000. Specific protein bands were detected using Odyssey® CLx Imaging System (LI-COR, USA).