Ab23841

Adipose tissues were fixed in 10% neutral formalin, embedded in paraffin, cut into 5 μm sections, and stained with UCP1 antibody (1:150, abcam, ab10983) to detect UCP1 protein expression and beige adipocyte formation as we described previously described (Zha et al. 2015; Nguyen et al. 2017). Tyrosine hydroxylase (TH) and UCP1 protein expression in IWAT and IBAT was measured by immunoblotting as we described (Zha et al. 2015; Nguyen et al. 2017). Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer containing 50 mmol/L Tris‐HCl, 1 mmol/L EDTA, 1% Nonidet P‐40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 200 mmol/L Na3VO3, 1% protease inhibitor mixture (Sigma), and 1% phosphatase inhibitor mixture (Sigma). Tissue lysates were separated using SDS‐PAGE. Proteins on the gels were transferred to nitrocellulose membrane (Bio‐Rad, Hercules, CA). The transferred membranes were blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680‐conjugated secondary antibodies (Life Science Techenologies). The blots were developed with a Li‐COR Imager System (Li‐COR Biosciences, Lincoln, NE). The following primary antibodies were used: UCP1 (1:500, abcam, ab23841), TH (1:1000, Millipore, AB152), and α‐Tubulin (1:1000, Advanced BioChemicals, ABCENT4777).

Adipose tissue was homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with a protease inhibitor mixture and phosphatase inhibitor mixture (Sigma, St. Louis, MO, USA). Tissue homogenates were separated by SDS-PAGE, which were transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), followed by blocking, washing, and incubating with primary antibodies and Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific, Waltham, MA, USA). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). The primary antibodies are UCP1 (1:500, Abcam, ab23841, Cambridge, MA, USA), TH (1:1000, Millipore, AB152, Burlington, MA, USA), phospho-HSL (pHSL) (1:1000, Cell Signaling, 4126, Danvers, MA, USA), CD36 (1:1000, Abnova, PAB 12463, Walnut, CA, USA) and α-Tubulin (1:1000, Advanced BioChemicals, ABCENT4777, Lawrenceville, GA, USA).