Secondary antimouse igg antibody

Whole protein lysates of the indicated samples were extracted by RIPA lysis buffer (JRDUN, Shanghai, China) with ethylene diamine tetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was estimated by using an enhanced bicinchoninic acid protein assay kit (Thermo Fisher, Cleveland, OH, USA). Equal amounts of total protein (25 μg) were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) overnight. After being blocked with 5% nonfat dry milk (P0216, Beyotime, Shanghai, China) for 1 h at room temperature, the membranes were probed at 4 °C overnight with the primary antibodies (ROCK1 [1:2000; Abcam, Cambridge, UK], ROCK2 [1:1000; Abcam, UK], DLC-1 [1:1000; Abcam, UK], β-catenin [1:5000; Abcam, UK], cyclin D1 [1:10,000; Abcam, UK], and GAPDH [1:2000; Cell Signaling Technology, Beverly, MA, USA]), and then the secondary anti-mouse IgG antibody (1:1000; Beyotime, Shanghai, China) for 1 h at 37 °C. An enhanced chemiluminescence system (Tanon, Guangzhou, China) was used to detect the protein expression value. The protein levels were normalized to GAPDH.