Anti mlkl

Whole-cell lysates were prepared using RIPA lysis buffer (Millipore, 20–188) and protein concentration was detected using a BCA protein assay kit (Thermo Scientific). A total of 150 μg of cell lysates was mixed with anti-RIP3 (2 μg, GeneTex, USA) or MagSi-protein A/G beads (50 μL, MagnaMedics, Netherlands) at 4°C overnight. The beads were then collected using a magnet for 2 min, washed with PBST washing buffer three times, and subjected to elution with 40 μL of 1x SDS loading buffer; the samples were incubated at 95°C for 10 min. Protein was separated using SDS-PAGE and transferred to 0.2 μM PVDF membranes (Bio-Rad, USA). Blots were then probed with anti-RIP1, anti-RIP 3, anti-MLKL (GeneTex), monoclonal anti-AIF (Cell Signaling), polyclonal anti-COX4 (GeneTex), polyclonal anti-PCNA (GeneTex), monoclonal anti-PARP1, monoclonal anti-PAR (Cell Signaling), anti-phospho-AMPKα (Thr172), anti-AMPKα, anti-phospho-p70 S6 kinase (Thr389), anti-p70 S6 kinase (Cell Signaling, USA), MitoProfile Total OXPHOS rodent antibody cocktail (MitoSciences, OR), and mouse anti-GAPDH (Abcam, USA). Signals were obtained using an enhanced chemiluminescence kit (Millipore) and densitometry was performed using Fusion-Capt software (Vilber Lourmat, Fusion FX7, France).

The following antibodies were used for Western blotting or immunostainings: anti-FLAG-HRP (Sigma-Aldrich; Cat. No. A8592, Lot. No. 116K6031), anti-RIPK1 (BD Bioscience, Cat. No. 610459), anti-RIPK3 (Abcam, Cat. No. ab56164), anti-MLKL (Genetex, Cat. No. GTX107538, Lot. No. LN40030), anti-p-MLKL (Abcam; Cat. No. 187091; Lot No. GR12667-11), anti-Histone (Santa Cruz; Cat. No. sc-8030, Lot No. L0808), anti-LDHA (Cell Signaling; Cat. No. 2012, Lot No. LN002), anti-Flotillin (Cell Signaling; Cat. No. 3253 S, Lot No. 10/2013), anti-tubulin-HRP (Abcam; Cat. No. ab21058, Lot. GR260583-2). For immunostainings, the following antibodies were used: anti-p-hMLKL S358 (Abcam; Cat. No. ab187091; Lot No. GR12667-11); anti-p-mMLKL S345 (Abcam; Cat. No. ab196436; Lot No. GR246882-1); anti-p-hRIPK3 S227 (Abcam; Cat. No. ab209384; Lot No. GR257915-8); anti-p-mRIPK3 T231 + S232 (Abcam; Cat. No. ab205421; Lot No. GR268165-4) or S232 (Abcam; Cat. no 195117; Lot No. GR268165-4). Full scans of all Western blots presented in figures or Supplementary Figures are shown in Supplementary Figs. 14–18.