Western Blot

Total protein was harvested from cells. Protein concentrations were determined by a BCA kit. An equal amount of protein was isolated with a 12% SDS-PAGE. The isolated protein was transferred onto PVDF membranes. After sealed with non-fat milk, the membranes were incubated with primary antibodies, such as anti-PDSS2 (ab251797, 1: 1000, Abcam, Cambridge, MA), anti-Nrf2 (ab137550, 1: 1000, Abcam), anti-PKCα (ab32376, 1: 1000, Abcam), anti-p53 (ab32389, 1: 1000, Abcam), anti-FSP1 (ferroptosis-suppressor-protein 1, ab197896, 1: 1000, Abcam), and anti-GAPDH (ab9485, 1:2500, Abcam) at room temperature for 2 hours. Then the membranes were incubated with secondary goat antirabbit antibody (ab6721, 1: 2000, Abcam). Finally, the bands were visualized with an enhanced chemiluminescent kit (Pierce) and analyzed with quantified LAS3000 imaging system (Fujifilm Corporation, Japan).