Anti phospho hormone sensitive lipase hsl

Cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer; 50 mM Tris−HCl, 150 mM sodium chloride (NaCl), 1% NP−40, 0.1% sodium dodecyl sulfate (SDS), a protease inhibitor cocktail, 50 mM sodium fluoride, and 0.2 M sodium orthovanadate). After centrifugation (12,000× g for 15 min), the protein concentration of the supernatant was measured. The proteins in the cell lysate were measured using the Braford assay. Next, 20 μg of protein from each sample was electrophoresed on 10% acrylamide SDS−polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Burlington, MA, USA). Membranes were then blocked in 5% skimmed milk for 1 h at room temperature and incubated with primary and secondary antibodies. The primary antibodies were anti−UCP1 (Abcam, Waltham, MA, USA), anti−PGC1α (Boster Bio, Pleasanton, CA, USA), anti−adipose triglyceride lipase (ATGL; Boster Bio), anti−phospho hormone−sensitive lipase (HSL; Cell Signaling, MA, USA), anti−TFAM (Invitrogen, Carlsbad, CA, USA), anti−NRF1 (Invitrogen), anti−AMPK (Cell Signaling), anti−phospho−AMPK (Cell Signaling), anti−AKT (Cell Signaling), anti−phospho−AKT, and β−actin (Sigma−Aldrich); proteins were visualized using chemiluminescent HRP substrate (Advansta Inc., San Jose, CA, USA).