Tina image software

Western blotting was performed as described previously^{46 (link)}. Blots were incubated overnight at 4 °C with polyclonal antibodies to periostin (Abcam, Cambridge, UK), fibronectin (Dako, Glostrup, Denmark), type I collagen (Southern Biotech, Birmingham, AL, USA), or β-actin (Sigma Chemical Co., Perth, Australia). The membranes were washed three times for 10 min in 1 × PBS with 0.1% Tween-20 and incubated in buffer A containing a 1:1000 dilution of horseradish peroxidase-linked goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). TINA image software (Raytest, Straubenhardt, Germany) was used to measure the band densities, and the changes in the optical densities of bands from the treated groups relative to control cells or tissues were used for analysis.

Total protein was extracted from the renal tissues and renal tubular epithelial cells using RIPA lysis buffer (Beyotime, China), and the concentration was estimated using a BCA Protein Assay kit (Beyotime, China). Equal amounts of protein (40 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes (PVDF). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against cyclin D1 (ab40754, Abcam, China), cyclin B (ab2096, Abcam, China), p21 (ab188224, Abcam, China), Bcl-2 (ab182858, Abcam, China), Bax (ab182733, Abcam, China), collagen I (ab34710, Abcam, China), collagen III (ab7778, Abcam, China) and Trib1 (ab137717, Abcam, China). GAPDH (ab8245, Abcam, China) served as the internal control. After three wash cycles, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies for 2 h at room temperature. Then, protein expression was visualized with an enhanced chemiluminescence reporter system detection kit. The band intensities were examined by TINA image software (Raytest, Straubenhardt, Germany) and normalized to that of GAPDH.