Oxphos cocktail

RIPA buffer (BR002, Biosolution) containing protease and phosphatase inhibitors was used to extract total proteins (P3100-001, P3200-001, GenDEPOT). The homogenates were centrifuged for 15 min at 13,000 rpm and 4 °C. A BCA protein assay kit was used to assess the protein concentration in the supernatants (23227, Thermo Scientific). Equal amounts of protein (20–40 μg) were resolved on SDS‒PAGE gels and then transferred to PVDF membranes. The following primary antibodies were used: UCP1 (ab10983, Abcam), OXPHOS cocktail (ab110413, Abcam), α-actin (A2066, Sigma), ATGL (PNPLA2) (#2439, Cell Signaling Technology), p-HSL (ser565) (#4137, CST), HSL (#4107, CST), p-Akt (#9271, CST) and Akt (#9272, CST). The membranes were subsequently treated with a corresponding secondary antibody of either anti-rabbit or anti-mouse IgG (horseradish peroxidase-linked) (PI-1000-1, Vector Laboratories). Enhanced chemiluminescence reagents (170-5061, Bio-Rad, Hercules) were used to visualize the bands, and the signals were evaluated using a Chemi-Doc XRS+ System (Bio-Rad). The concentration of the target protein was then compared to that of β-actin. ImageJ software (NIH) was used to measure band intensities.

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).

A list of plasmids is provided in Table S1. Plasmids were prepared by standard methods. Antibodies against the following proteins were purchased: UBQLN1/2 (Sigma, clone 5F5), UBQLN4 (Abcam ab106443), L9 and Tom20 (Santa Cruz Biotech., T-17 and FL-145), HA (Covance, clone 16B12), Rpt5 (Abcam ab22635), α7 (Enzo Life Sciences PW8110), FLAG (Sigma, clone M2), Hsc/Hsp70 (AssayDesigns, SPA-822), Hsp60 (Abcam ab46798), ClpP (Abcam ab124822), Actin-HRP (Sigma A3854), OxPhos cocktail (Abcam ab110411). FLAG-M2 Affinity resin was from Sigma, and GFP-trap from Chromotek. Antibodies to Bag6, TRC40, SGTA, TRAPα, GFP, and RFP have been described (Fons et al., 2003 (link), Mariappan et al., 2010 (link), Hessa et al., 2011 (link)). Anti-Myc was clone 9E10. Anti-HA used for IPs and blots (e.g., Figure 2A) was raised in rabbits against the KLH-HA peptide conjugate. Recombinant proteins were either purchased (His6-Ubiquitin, E1, and E2 from Boston Biochem) or expressed and purified from E. coli as described (Mateja et al., 2015 (link)).