Sc 7947

Homogenized tissue or cells were lysed with ice-cold RIPA buffer (Pierce, 89900) containing 1X protease inhibitor cocktail (GenDEPOT, P3100) and incubated at 4 °C for 30 min. After centrifugation at 15,000 × g for 15 min, the supernatant was moved to a new tube. Protein concentrations were measured using the Bradford assay (Bio-Rad, 5000001). Protein samples were directly analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with SDS sample buffer (60 mM Tris-Cl (pH 6.8), 10% sodium lauryl sulfate, 25% glycerol, 100 mM dithiothreitol, 0.04% Bromophenol blue) without boiling. Western blot analysis was performed according to standard methods. Antibodies used in immunoblot analyses included those against LETMD1 (LSBio, LS-C384640, 1:1000); UCP1 (Abcam, ab10983, 1:1000); HSP90 (Santa Cruz, sc-7947, 1:2000); α-Tubulin (Sigma-Aldrich, sc-8035, 1:3000); OXPHOS cocktail (Abcam, ab110413, 1:1000); Streptavidin-HRP (Thermo Scientific, S911, 1:3000); and Flag (Sigma-Aldrich, F3165, 1:3000). The specific signals were amplified by horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody (Santa Cruz, 1:3000).

Cells were lysed in RIPA buffer supplemented with Complete Protease Inhibitor Cocktail and PhosphoStop (both from Roche, Mannheim, Germany) and Western blot analyses were performed as described previously (10 (link)). Primary antibodies were purchased from: Cell Signaling, Frankfurt, Germany [anti-ERK1/2 (9102), anti-phospho-ERK1/2 (9106), anti-TRAIL-R4 (8049), anti-TRAIL-R2 (3696), anti-mouse-IgG-HRP (7076), anti-rabbit-IgG-HRP (7074)]; EMD Millipore Corp., USA [TRAIL-R1 (AB16955)], BD Biosciences, USA [anti-COX-2 (610203)], TRAIL-R3 (DcR1, im-245-1, Imgenex, San Diego, USA), HSP90α/β (Santa Cruz, sc-7947), and from Sigma-Aldrich [anti-β-actin (A5441)].