Ab10527

Primary antibodies used in this study were: rat anti-MLKL (clone 3H1, produced in-house^{1 (link)}; available as MABC604, EMD Millipore, Billerica, MA, USA; 1:1000), rabbit anti-MLKL phospho-S358 (AB187091, Abcam; 1:4000), anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:2000), anti-Actin (A-1987, Sigma-Aldrich, St Louis, MO, USA; 1:3000), and anti-VDAC1 (AB10527, EMD Millipore; 1:5000). Recombinant hTNF-Fc, produced in-house, and the Smac mimetic, Compound A, have been previously described^{38 (link),39 (link)}. The pan-caspase inhibitor, IDN-6556/emricasan, was provided by Tetralogic Pharmaceuticals.

Interscapular BAT and GM from male TRPV1 WT and KO mice and WT mice administered with AMG9810 or vehicle were immediately dissected and snap frozen in liquid nitrogen for mitochondrial protein extraction using a Mitochondria Isolation Kit (Thermo Scientific) according to the manufacturer’s instructions. Equal amounts of protein (15–30 μg) were separated by SDS-PAGE under reducing conditions in 10% polyacrylamide gels. The proteins were transferred to PVDF membranes (EMD Millipore, Feltham, United Kingdom), blocked in 5% nonfat dried milk (1 h at room temperature), and incubated with primary antibody for UCP1 and UCP3 (1:1000 ab23841 and ab3477; Abcam, Cambridge, United Kingdom) at 4°C for 12 h. The voltage-dependent anion-selective channel (VDAC) was used as a loading control (1:1000 AB10527; EMD Millipore). The membranes were exposed under ECL (Luminata Classico Western HRP substrate; EMD Millipore) in a G-BOX Gel Documentation System (Syngene, Cambridge, United Kingdom) using the manufacturer’s software (Syngene 2D gel imaging software; Syngene). Samples were quantified densitometrically using ImageJ (National Institutes of Health, Bethesda, MD, USA).