Rabbit monoclonal antibody against α sma

After lysis and denaturation of HK2 cells or kidney tissues from each group using radioimmunoprecipitation assay (RIPA) buffer, the proteins (50 µg) were separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose (NC) membranes. After blocking with 1 × casein for 1 h to prevent non-speci c binding, NC membranes were incubated with the following primary antibodies at 4 °C for overnight: (a) Rabbit monoclonal antibody Ecadherin (BD Bioscience, USA) diluted to 1:100; (b) rabbit monoclonal antibody against α-SMA (Abcam, UK) diluted to 1:300; (c) mouse monoclonal antibody against CTGF (Abcam) diluted to 1:200; (d) rabbit polyclonal antibody (Proteintech, USA) against bronectin diluted to 1:500; (e) rabbit polyclonal Col3A1 antibody (Proteintech) diluted to 1:500; (f) mouse monoclonal β-actin antibody (Beyotime, China) diluted to 1:10000. The membranes were washed with TBST (Tris buffered saline, with Tween-20, 20 mM of Tris, 140 mM of NaCl, and 0.1% Tween-20) and probed with 1:1000 diluted secondary antibodies at room temperature (25℃) for 2 h. Enhanced chemiluminescence (ECL) western blotting kit (APPLYGEN, China) was used to detect target bands, and β-actin was used as internal reference to calculate the relative expression of protein in each experimental group.