Immunoblotting of Necroptosis Mediators

Primary antibodies used in this study were: rat anti-human RIPK3 (clone 1H2, produced in-house; 1:1000; available as MABC1640, EMD Millipore, Billerica, MA, USA) [25 (link)], rat anti-mouse RIPK3 (clone 1H12, produced in-house; 1:1000) [41 (link)], rabbit anti-human RIPK3 phospho-S227 (D6W2T, CST, 1:2000), rabbit anti-human RIPK3 phospho-S227 (EPR9627, Abcam; 1:2000) rat anti-human MLKL pseudokinase domain (clone 7G2, produced in-house; 1:2000 dilution; available as MABC1636, EMD Millipore, Billerica, MA, USA) [27 (link)], rat anti-human MLKL (3H1, produced in-house; 1:1000; available as MABC604, EMD Millipore, Billerica, MA, USA) [25 (link)], mouse anti-human RIPK1 (610459, BD Transduction Laboratories; 1:1000), rabbit anti-human MLKL phospho-S358 (AB187091, Abcam; 1:2000), mouse anti-GAPDH (MAB374, Millipore; 1:2000) and anti-Actin (C4) HRP (sc-47778 HRP, Santa Cruz Biotechnology; 1:10,000). The Smac mimetic, Compound A [42 (link)] and the pan-caspase inhibitor, IDN-6556/Emricasan, were provided by Tetralogic Pharmaceuticals, and recombinant hTNF-Fc [43 (link)] was produced in-house.