Chemiscope 3300 mini imaging system

Renal tissues or cell samples were lysed on ice with lysis buffer containing cocktail proteinase inhibitors and protein phosphatase inhibitors (Thermo Fisher, Rockford, IL, USA). The extracted protein concentrations were quantified via BCA assay, mixed with loading buffer and boiled. Proteins were resolved on 10% SDS-PAGE in running buffer, transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, USA), blocked with blocking buffer, and blotted with the primary antibodies at 4℃ overnight. Blots were washed and followed by incubation with secondary antibodies. After washing three times with PBST, signals were detected by chemiluminescence (Tanon, Shanghai, China), and visualized by ChemiScope 3300 Mini Imaging System (CLiNX, Shanghai, China). The western blot data was quantified by IMAGE J.

Total cell extracts were lysed on ice using a lysis buffer (containing proteinase and phosphatase inhibitors; Beyotime Biotech Co., Ltd.). Protein concentrations were measured using the BCA Kit (Vazyme Biotech Co., Ltd.). Samples were mixed with SDS-PAGE Sample Loading Buffer (5X, Beyotime Biotech Co., Ltd.) and denatured at 95°C for 5 min, then resolved on 10% gels using SDS-PAGE in running buffer (Sangon Biotech Co., Ltd. Tris, 3 g; Glycine, 14.4 g; SDS, 1 g; Fix the volume to 1 liter with DD water.). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk at room temperature for 1 h, the PVDF membranes were incubated with primary antibodies at 4°C overnight, washed with PBST and then incubated with secondary antibodies at room temperature for 1 h. After washing with PBST 3 times, signals were detected using an enhanced chemiluminescence system (Vazyme Biotech Co., Ltd.) and acquired by ChemiScope 3300 Mini Imaging System (Clinx Science Instruments Co., Ltd.).