Enhanced chemiluminescence western detection reagents

The total protein was extracted from the samples using radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor tablet (Roche). Protein concentrations were quantified using a Bicinchoninic Acid Protein Assay kit (Biosky Biotechnology, Nanjing, China) according to the manufacturer’s protocol. Thirty micrograms of protein samples were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to PVDF membranes (0.45 µm; EMD Millipore, Billerica, MA, United States). Membranes were blocked for 1 h at room temperature with 5% BSA (Sangon Biotech Co., Ltd.) in TBS-Tween-20 (containing 5 mM Tris-HCl (pH 7.6), 136 mM NaCl and 0.05% Tween-20). Then Membranes were incubated with anti-TBP (Proteintech, China, 22006-1-AP, 1:1,000), anti-MKL1 (Proteintech, China, 21166-1-AP, 1:1,000) overnight at 4°C. Subsequently, the primary antibodies were detected with secondary antibody (anti-Rabbit, Beyotime Institute of Biotechnology, China, A0208, 1:3,000) and scanned by using enhanced chemiluminescence western detection reagents (Thermo Fisher Scientific, Inc.). Image-Lab version 5.2.1 software (Bio-Rad Laboratories, Inc.) was used to quantify the intensity of the band in western blot assay.