Ecl plus

Kidney tissues were lysed in radioimmunoprecipitation (Beyotime) assay lysis buffer containing phenylmethylsulfonyl fluoride (Solarbio). Protein concentrations were determined using a bicinchoninic acid assay. Protein samples were separated on a 30% sodium dodecyl sulfate–polyacrylamide gel at 120 V and then transferred to nitrocellulose membranes (Pall Corporation) using a Turbo Transfer System (Bio‐Rad). Membranes were blocked for 1 h at 25 °C and then incubated with the appropriate primary antibodies at 4°C overnight, followed by washing three times with Tris‐buffered saline‐Tween 20 (Sigma Aldrich). Membranes were then incubated with horseradish peroxidase‐conjugated secondary antibodies for 2 h at 25°C and washed with Tris‐buffered saline‐Tween 20. Protein bands were visualized using chemiluminescence with ECL Plus (#P1050; Applygen) according to the manufacturer's instructions. The primary antibodies used in our study were as follows: TGF‐β1 (1:1000, #21898; Proteintech), α‐SMA (1:1000, #55135; Proteintech), Smad3 (1:1000, #ab52903; Abcam), p‐Smad3 (1:1000, ab40854; Abcam), and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; 1:1000, #10494‐1‐AP; Proteintech).

The proteins (20 μg) were separated by 8% SDS/PAGE and then electroblotted onto a PVDF membrane (Millipore), which was then washed for 10 min with TBST and immersed in blocking buffer containing 5% nonfat dry milk in TBST for 1 h at 25 °C. The blot was washed with TBST and then incubated with a rabbit polyclonal primary GAP43 antibody (Abcam, 1:5000 ab75810) overnight at 4 °C. After the blot was washed in TBST, it was incubated with a secondary antibody against rabbit IgG (Santa Cruz 1:2000) for 1 h at 25 °C. The blot was finally washed with TBST, and the protein bands were visualized with a chemiluminescence system (ECLPlus, Applygen Technologies, Inc.).