Anti p mlkl

For flow cytometry, we used anti-CD45, anti-CD11b, anti-MHCII, anti-CD11c, anti-F4/80, anti-TNFR1, anti-B220, anti-CD3, anti-CD4, anti-CD8 and anti-NK1.1 antibodies (all from BioLegend). For Western blotting, anti-MLKL (1:1000), anti-cleaved caspase 3 (1:1000), anti-cleaved caspase 8 (1:1000), anti-TNFRI (1:1000), β-actin (1:1000) and HRP conjugated secondary antibody (1:5000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-pMLKL (1:1000) was obtained from Sigma Aldrich (St. Louis, MO, USA). For confocal microscopy, we used anti-F4/80 (Abcam, Cambridge, UK), Anti-pMLKL (Sigma Aldrich), anti-cleaved caspase 3 and anti-TNFR1 (Cell Signaling Technologies). Secondary antibodies (goat antirat IgG [H+L], Alexa 647 [catalog A21247]; goat antirabbit IgG [H+L], Alexa Fluor 488 [catalog A11008]; and goat anti-mouse IgG [H+L], Alexa Fluor 594 [catalog A11032]) were obtained from Invitrogen (Waltham, MA, USA), and Fluoroshield mounting medium with DAPI (Abcam, catalog ab104139) was used to stain nuclei. For neutralization studies, an anti-TNFR1 antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Streptozotocin (STZ), Nicotinamide (NA), deoxyadenosine monophosphate, acetyl choline, zVAD-FMK, Nec-1 and shikonin were obtained from Millipore Sigma (St. Louis, MO, USA). Pyridoxine and 2-Ketohexanoic acid were purchased from Cayman Chemicals.

RIPA Lysis Buffer (Beyotime, China) containing PMSF (Beyotime, China) and PhosSTOP (Sigma-Aldrich, USA) was used to lyse the cells with indicated treatment. After quantification, the protein samples were separated by SDA-PAGE gel (Beyotime, China). Primary antibodies (Anti-RIPK1, BD Biosciences, USA; Anti-RIPK3, Abcam, USA; p-RIPK3, Abcam, USA; Anti-MLKL, Sigma-Aldrich, USA; Anti-p-MLKL, Sigma-Aldrich, USA; Anti-GAPDH, Beyotime, China) were used at 4 °C overnight. The protein bands were detected by ECL Kit (BOSTER, China).