Chemiluminescence system

Radio-immunoprecipitation assay buffer (Beyotime, Haimen, China) was used to extract the fragments of renal tissue and cellular proteins. The extracted protein concentrations were quantified by the bicinchoninic acid reagent (Beyotime, Haimen, China). The samples (40 μg) were separated via 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to nitrocellulose membranes. The membranes were sealed with 5% skimmed milk for 1 h, washed with phosphate-buffered saline (PBS) three times, and incubated with applicable primary antibodies overnight. They were then incubated with the secondary antibody (Rockland Immunochemicals Inc., PA, USA) for 30 min. The band intensities were exposed using a chemiluminescence system (Bio-Rad, Hercules, CA, USA). Image J software (NIH, Bethesda, MD, USA) was used to quantitatively analyze the protein bands after normalization with β-actin.

Western blotting was performed as previously described [31 (link)]. Primary antibodies against mTOR (1 : 1000; Cell Signaling Technology), ULK1 (1 : 500; Proteintech, Wuhan, China), p-ULK1 (1 : 2000; Proteintech), ATG13 (1 : 500; Servicebio, Wuhan, China), Beclin1 (1 : 1000; Proteintech), p62 (1 : 500; Servicebio), LC3 (1 : 1000; Servicebio), MMP13 (1 : 500; Servicebio), COL2A1 (1 : 1000; Abcam, Cambridge, UK), and GAPDH (1 : 3,000; Cell Signaling Technology) were used. Protein bands were detected using a chemiluminescence system (Bio-Rad Laboratories, Hercules, CA, United States) with an enhanced chemiluminescence (ECL) kit (Millipore, Darmstadt, Germany). Signal intensity was compared using the ImageJ software (NIH, Bethesda, MD, USA).

Whole cells were lysed in cell lysate buffer (PMSF:RIPA=1:100, Beyotime, China), and protein concentrations were quantified with a BCA protein quantification kit (Beyotime, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in fat-free milk and incubated with specific antibodies at 4 °C for 12 hours. The antibodies included anti-PHLPP2 (Abcam, USA), anti-CD133 (Proteintech, USA), anti-CD44 (CST, USA), anti-EPCAM (CST, USA), anti-Nrf2 (CST, USA), and anti-GAPDH (Hangzhou Xianzhi, China). Membranes were then incubated with secondary antibody (ZSGB-bio, China) and detected using a chemiluminescence system (Bio-Rad).

Total cellular proteins were lysed by RIPA buffer containing protease inhibitors (Sigma, USA). The protein extractions were harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extractions were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After the incubation with a high affinity anti-METTL3 antibody (1:1000, Abcam, USA), anti-DGCR8 antibody (1:1000, Abcam, USA), anti-PTEN antibody (1:1000, Abcam, USA), anti-β-actin antibody (1:1000, Cell Signaling Technology, USA) or anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA), the membranes were then incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). After washes, signals were detected using a chemiluminescence system (Bio-Rad, USA) and analyzed using Image Lab Software.