Anti ucp1 ab10983

EV pellets secreted by 1 g of VAT, SAT, and BAT explants were lysed, resuspended in Laemmli sample buffer, and processed for immunoblotting, as previously described (Camino T, Lago-Baameiro N; Bravo SB, Molares-Vila A, Sueiro A, Couto I, Baltar J, Casanueva FF, n.d.) [6 (link)]. Primary anti-Alix (sc-53538, dilution 1:500), anti-CD81 (sc-166029, dilution 1:500), anti-ceruloplasmin (sc-365205, dilution 1:500), anti-fatty acid synthase (sc-48357, dilution 1:500), and anti-CD14 (sc-515785, dilution 1:500) were purchased from Sta. Cruz Biotechnology (Santa Cruz, CA, USA); anti-Perilipin1 (PA5-55046, dilution 1:1000), anti-Vimentin (PA5-27231, dilution 1:1000), and anti-FABP4 (PA5-19811, dilution 1:1000) were purchased from ThermoFisher Scientific S.L (Waltham, MA, USA); anti-ACLY (4332s) was purchased from Cell Signaling Technology (Danvers, MA, USA); and anti-UCP1 (ab10983) was purchased from Abcam (Cambridge, UK).

White adipose tissue was homogenized in lysis buffer (50 mM HEPES, PH 7.4, 150 mM NaCl, 1 % Triton X-100, 5 mM EGTA) and homogenates were spun at 20,000 g. Supernatants were extracted with 4 volumes of cold acetone and the precipitated protein was resuspended in 10 % SDS, made up to a final SDS concentration of 2 % with water, and spun at 12,000 g for 2 min to obtain the final supernatant. Protein was determined by the BCA protein assay (Thermo Fisher, Rockford, IL). Proteins were separated in 10 % Bis-Tris NuPAGE gels and transferred to PVDF membranes employing the iBlot2 (Thermo Fisher, Rockford, IL). Membranes were blocked and incubated with primary and secondary antibodies in Odyssey blocking buffer (Li-Cor, Lincoln, NE). Primary antibodies were: anti-FGF21, AF3057, and anti-CD137, AF937 (R&D Systems, Minneapolis, MN), anti-P2RX5, sc-398682 (Santa Cruz Biotechnology, Dallas, TX), anti-beta-actin, sc-47778 (Santa Cruz, Dallas, TX), and anti-UCP1, ab10983, (abcam, Cambridge, MA). Dye-conjugated secondary antibodies were from Li-Cor (Lincoln, NE). The bands were detected by immunofluorescence.

Proteins from BAT, scWAT, and pgWAT were extracted using mammalian protein extraction reagent (MPER, Pierce) supplemented with halt phosphatase and halt protease inhibitors (Pierce) as per the manufacturer's instructions, separated by gel electrophoresis and analyzed using the antibodies anti‐CDK4 (C‐22, sc‐260, Santa Cruz), anti‐p‐CREB Ser133 (87G3, 9198S, Cell Signaling Technology), anti‐CREB total (48H2, 9197S, Cell Signaling Technology), anti‐α‐tubulin (DM1A, Sigma), anti‐UCP1 (ab10983, Abcam), anti‐HSP90 (4874S, Cell Signaling Technology), anti‐GAPDH (6C5, sc‐32233, Santa Cruz), and anti‐TH (AB154, Millipore).