Rabbit anti smad2 3

Podocyte samples were treated as described above for qRT-PCR. Total protein was extracted from each sample to prepare cell lysates (Sangon, Shanghai, China) and stored at -20°C. The bicinchoninic acid (BCA) protein quanti cation method was used to ensure that the concentration of each sample was basically equal. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes with electrophoresis systems (Tanon VE180 and Tanon VE186, Shanghai, China). The PVDF membranes were blocked with 5% (w/v) skimmed milk powder for 2 h and incubated at 4°C overnight with the following primary antibodies (1:1000): rabbit anti-Smad2/3, p-Smad2/3, Smad7 and NOX4 (Abcam, UK). After being washed with 1× PBS solution (Sangon, Shanghai, China) three times, the membranes were incubated with HRP-labelled goat anti-rabbit IgG secondary antibodies (1:5000) (Abcam, UK). Immunoreactivity was determined with enhanced chemiluminescence (ECL) reagent (Thermo Fisher, US). A gel imaging system (BIO-RAD Gel Doc XR+, US) and software (BIO-RAD Image Lab Software, Version 5.1 and SPSS 20.0) were used for imaging and statistical analysis. GAPDH was used as an internal control to ensure equal protein loading.