Mitochondrial total oxphos protein antibody set

Immunoblotting for protein detection was conducted as we described [27 (link),32 (link)]. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Tissue lysates were resolved by SDS-PAGE gels. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). Primary antibodies used were as follows: Anti-UCP1 antibody (1:500, ab23841, Abcam, Cambridge, MA, USA); Anti-α-Tubulin antibody (1:1000, ABCENT4777, Advanced BioChemicals, Lawrenceville, GA, USA); Mitochondrial total OXPHOS protein antibody set (Abcam,ab110413); and Anti-pHSL (4126s, Cell Signaling Technology, Danvers, MA, USA); DNMT3b (sc-393845, Santa Cruz, Dallas, TX, USA, sc-393845); HSL (Cell Signaling, 4107s).

Western blot was conducted to assess the protein of interest [23 (link)]. Snap-frozen samples were homogenized in a modified radioimmunoprecipitation assay buffer and followed by centrifugation to extract supernatants. An equal amount of protein was loaded on SDS-PAGE, which was transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The proteins were then immunoblotted with various primary and secondary antibodies, and visualized on a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). The protein antibodies are listed as follows: UCP1 antibody (1:500, abcam, ab23841), Mitochondrial total OXPHOS protein antibody set (Abcam, ab110413), phospho-HSL antibody (pHSL) (1:1000, Cell Signaling 4126), HSL antibody (1:1000, Cell Signaling 4107), α-Tubulin antibody (1:1000, Advanced BioChemicals, ABCENT4777, Lawrenceville, GA, USA) and the secondary antibody Alexa Fluor 680 (ThermoFisher Scientific).