Ab118470

Briefly, cells were scraped into ice-cold radioimmunoprecipitation assay (RIPA) buffer and 1% Halt protease and phosphatase inhibitor cocktail. Briefly, cells were scraped into ice-cold radioimmunoprecipitation assay (RIPA) buffer and 1% Halt protease and phosphatase inhibitor cocktail. Cell lysates were centrifuged at 14,000 rpm for 15 min at 4°C, and supernatants were collected. Protein concentrations were determined using the Bradford method. Cellular proteins were separated by 10% SDS-PAGE, transferred onto PVDF membranes (0.45 μM, IPVH00010, Millipore), and blocked with 5% skim milk at room temperature for 1 h. Membranes were incubated with the following primary antibodies: anti-YAP1 Polyclonal Antibody (PA5-87568, Invitrogen, 1:2000), anti-ALOXE3 (ab118470, Abcam, 1:1000), anti-β-actin (ab8226, Abcam, 1:3000) and anti-GAPDH (60004-1-Ig, Proteintech, 1:5000) overnight at 4°C. They were washed and incubated with the horseradish peroxidase conjugated anti-rabbit or mouse IgG (H + L) (1:3000; Proteintech). The enhanced chemiluminescence solution (GE Healthcare) was used for visualizing protein expression.

To detect the protein level alteration of ferrotosis-related molecules, western blotting was performed. The cell samples were lysed by RIPA buffer containing 0.1% SDS. After running the SDS–PAGE, the protein samples were transferred onto the PVDF membrane, and followed by antibody incubation. The antibodies were listed as follows: rabbit anti-SLC7A11 (1:1000, PA1-16893, Invitrogen, USA), rabbit anti-ALXOE3 (1:800, ab118470, Abcam, USA), and mouse anti-β-actin (1:5000, A5441-100UL, Sigma, USA). The blots were explored with ECL, and the immunoreactive signals were generated in a luminescence detection system using horseradish peroxidase-labeled secondary antibody.