2.13 Western blot assay
Fresh cells or kidney tissues were collected and lysed on ice for 30 minutes. Equal amounts of protein (20 µg) were separated by 10-15% SDS-PAGE, and the separated proteins were transferred onto PVDF membranes and blocked with 5% nonfat powder milk in PBS buffer for 1 h at room temperature. The cells were incubated with primary antibodies against β-actin, KIM1, NGAL, GPx4, xCT, FTL, FTH-1, TFR, Nrf2, HO-1 and NQO1 overnight at 4°C and then with an HRP-conjugated secondary antibody (1:5000) at room temperature for 1 h. The relative expression levels were determined with Image J software.