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.