Mab5457

For protein blot analyses, cells were grown to full confluence, washed twice with 1X PBS, immediately scraped off the plate into a microcentrifuge tube with 1X Laemmli sample buffer for CISD2 and mNT antibodies, and heated to 95°C for 10 minutes. For GPX4, TfR, TXNIP and TRX2, RIPA (Radioimmunoprecipitation assay; Sigma-Aldrich) buffer was used and lysates were kept on ice for 2 hours with vortex every 30 minutes, then centrifugation at 15,000 rpm for 15 minutes, supernatant was used for protein denaturation with Laemmli sample buffer 5X. Protein gels were loaded with equal amounts of protein, determined using The Pierce 660 nm Protein Assay (Thermo Scientific™, Cat. No. 22662) [11 (link)], and analyzed using antibodies against CISD2 and mitoNEET [23 , 33 (link)], TXNIP (Novus Biologicals, NBP2–27095), Thioredoxin 2 (Novus Biologicals, NBP1–92499), β-actin (R&D Systems, MAB8929), GPX4 (R&D Systems, Cat. No. MAB5457), and TfR (Abcam, ab84036). Goat Anti-Rabbit IgG, H & L Chain Specific Peroxidase Conjugate (Sigma, 401315) and Peroxidase-conjugated AffiniPure Goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, AB_10015289) were used as secondary antibodies (7, 24). All experiments were repeated at least 3 times.