Phosphorylated mlkl

Whole-cell lysates were obtained by lysing the cells in RIPA lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS]) supplemented with phosphatase inhibitors (5 mM sodium fluoride, 1 mM sodium orthovanadate and 1 mM sodium pyrophosphate) and protease inhibitor cocktail (Thermo Fisher Scientific). The boiled lysates were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific). The membranes were blocked with 5% non-fat dried milk and incubated with the following specific primary antibody: caspase-3 (Abcam), caspase-8 (Cell signalling technology), caspase-9 (Cell signalling technology), FASLG (Abclonal), TRAIL (Abcam), TNF-α (Cell signalling technology), PUMA (Abcam), MLKL (Millipore), phosphorylated MLKL (Abcam) followed by HRP-conjugated goat anti-mouse, rabbit or rat secondary antibodies (Thermo Fisher Scientific) and WesternBright ECL solution HRP substrate (Advansta, CA, USA). The membranes were stripped and re-probed with anti-β-actin antibody (Sigma) as internal control for protein loading.

Cells were lysed using RIPA buffer supplemented with aprotinin (Sigma), and Halt Protease Inhibitor Cocktail (100X) (Thermo Scientific). Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad, Mississauga, ON). Primary antibodies used in this study include caspase-1 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-1 (AdipoGen Life Science, San Diego, CA), caspase-3 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-3 (Cell signaling technology, Danvers, MA), HMGB1 (Cell signaling technology, Danvers, MA), MLKL (Abcam, Boston, MA), phosphorylated MLKL (Abcam, Boston, MA), CRT (Abcam, Boston, MA), and GAPDH (Sant Cruz Biotechnology, Dallas, TX). The expression levels Relative expression of proteins were visualized using the corresponding secondary HRP-conjugated antibodies and Amersham ECL select western blotting detection reagent, as described previously (46 (link)).

The antibodies used in immunofluorescence and western blot included Ninj2 (R&D systems, Cat# AF5056), Cleaved-Caspase 3 (Cell Signaling Technology, Cat# 9661), phosphorylated MLKL (abcam, Cat# ab196436), Laminin 211 (abcam, Cat# ab7463), Lama2 (Boster, Cat# BA3201-2), Sox10 (abcam, Cat# ab155279), Sox10 (abcam, Cat# ab216020), Mpz (abcam, Cat# ab31851), Neurofilament (abcam, Cat# ab8135), Ki67 (Gene Tex, Cat# GTX16667), phosphorylated FAK (abcam, Cat# ab4792), GAPDH (Proteintech, Cat# 60004-1-lg), HA (Santa Cruz, Cat# sc-7392), and FLAG (sigma, Cat# B3111).