Epncir144

BMDMs were lysed with cell lysis buffer (Cell Signaling) supplemented with 2 mM PMSF (Sigma-Aldrich) and a complete Ultra protease inhibitor cocktail (Roche). Protein concentrations were determined by using Pierce 660-nm Protein Assay Reagent (Thermo Fisher Scientific) and normalized, and the protein samples were denatured by adding reducing buffer (Thermo Fisher Scientific) before incubation at 95°C for 5 min. Samples were separated in Mini-Protean TGX gels (Bio-Rad) and then transferred to the nitrocellulose membrane by using Trans-Blot Turbo Transfer System Bio-Rad machine according to manufacturer’s instructions. Nitrocellulose membranes were blocked by using Odyssey blocking buffer (LI-COR) at room temperature for 2 h. Immunoblotting was performed by adding either anti-Gpx4 (1:1,000, clone EPNCIR144; Abcam) or anti-β actin (1:1,000, clone 8H10D10; Cell Signaling) and the membranes incubated at 4°C overnight. Subsequently, membranes were washed with 0.02% Tween20 (Sigma-Aldrich) in 1× DPBS and then treated with the respective secondary detection reagent (IRDye 800CW Donkey anti–rabbit IgG [LI-COR] or IRDye 680CW Donkey anti–mouse IgG [1:10,000; LI-COR]) at room temperature for 1 h. After washes in 0.02% DPBS-Tween20, membranes were imaged on a LI-COR Odyssey.

Infiltrating cells in the lungs were stained by using appropriate combinations of directly conjugated or secondary antibodies. Antibodies used were directed against Ly6C (clone HK1.4), Ly6G (clone 1A8), CD11c (clone N418), F4/80 (clone BM8), CD45.2 (clone 104), CD45 (clone 30-F11), CD11b (clone M1/70), Syglec F (clone E50-2440), CD4 (clone GK1.5), CD8 (clone 53–6.7), Gpx4 (monoclonal rabbit unconjugated, clone EPNCIR144; Abcam). Unconjugated monoclonal rabbit antibody was detected with donkey F(ab′)2 anti–rabbit IgG H&L pre-adsorbed (ab181347; Abcam), and rabbit IgG monoclonal (ab172730; Abcam) was used as primary isotype control. Live/Dead fixable blue dead cell stain kit and Fixable Viability Dye eFluor 780 dye were purchased from Invitrogen Molecular Probes and eBioscience, respectively, and the staining was performed according to the manufacturer’s specification. All samples were acquired on LSR Fortessa flow cytometer (BD Biosciences) and analyzed by using FlowJo 10.4.2 software (Tree Star).