Sc 8035

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).

The custom recombinant antibody-like MAR binding reagent (anti-MAR) was generated and purified in-house (now available from Millipore Sigma, MABE1076; RRID:AB_2665469) (Gibson et al., 2017 (link)). The antibodies used were as follows: PARP-7 (Invitrogen, PA5-40774; RRID:AB_2607074), α-tubulin (Santa Cruz, sc-8035; RRID:AB_628408), β-tubulin (Abcam, ab6046; RRID:AB_2210370), SNRP70 (Abcam, ab51266; RRID:AB_10673827), Flag (Sigma-Aldrich, F3165; RRID:AB_259529), HA (Sigma-Aldrich, H3663; RRID:AB_262051), rabbit IgG fraction (Invitrogen, 10500C; RRID:AB_2532981), goat anti-rabbit HRP-conjugated IgG (Pierce, 31460; RRID:AB_228341), goat anti-mouse HRP-conjugated IgG (Pierce, 31430; RRID:AB_228307), and Alexa Fluor 488 goat anti-mouse IgG (ThermoFisher, A-11001; RRID:AB_2534069).