Western Blot Analysis of Antioxidant Proteins

We performed Western blot, as previously recorded ((Zhang et al., 2016 (link); Fei et al., 2020 (link))). The proteins extracted from renal tissues or cells were lysed in a RIPA buffer containing protease and phosphatase inhibitors, and the protein content was detected by using the BCA protein assay kit (Beyotime). Protein samples (50 ug) from each group were electrophoresed in 10% SDS-PAGE gel and then transferred to a PVDF membrane. After blocking with 5% nonfat dry milk, the blots were incubated with the primary antibodies against GPX4 (1: 1000, DF6701, Affinity), ACSL4 (1: 1000, A14439, Ablonal), Nrf2 (1: 1000, AF7904, Affinity), SIRT-1 (1: 1000, DF6033,Affinity), and or β-actin (1: 200, ab181602, Abcam) overnight at 4°C. Subsequently, the blots were washed in TBST and incubated with a secondary antibody for 2 h at room temperature. The bands were determined with an ECL system applying an ECL kit (Applygen), and band intensities were checked by BandScan software.