Mab6158

Immunoblotting was performed as described [14 (link)] with the following commercial primary antibodies: SMAD 1/5/9 (ab66737, Abcam, Cambridge, UK), total OXPHOS Antibody Cocktail (ab 110413, Abcam, Cambridge, UK), UCP1 (MAB6158, R&D System), β-tubulin (#2128), pSMAD 1/5/9 (#13820), Akt (#9272) and phospho-Akt (ser 473) (#9271) (all from Cell Signalling Technology, Danvers, MA, USA) and phospho-Akt (thr308) (#9275S BioLabs). Quantifications were performed by normalisation against loading controls.

Adipose tissues were stained with hematoxylin and eosin (HE) according to the standard protocols. For immunohistochemistry, rabbit anti-UCP1 antibody (Alpha Diagnostic, ucp1-a, diluted 1:400) or rabbit anti-IRX3 (Abcam, ab25703, diluted 1:1000) was used to incubate the sections, followed by incubation with HRP-linked secondary antibody (Dako, k500711). Electron microscopy of browning adipose tissue was performed as previously described (Wang et al., 2013 (link)). To determine the localization of IRX3 in the browning adipocytes, beige cells after eight days induction were fixed and incubated with rabbit anti-IRX3 antibody (Abcam, ab25703) and mouse anti-UCP1 antibody (R&D, MAB6158), followed by incubation with Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (ThermoFisher, R37117), 488–conjugated goat anti-mouse secondary antibody (ThermoFisher, A32723), and DAPI (SouthernBiotech, 0110-20). Images were acquired with a confocal laser-scanning microscope (Zeiss). All the images were representative of three independent experiments.

Sample protein extracts were obtained from cell monolayers and were separated using SDS-PAGE under reducing conditions and then transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). For immune-detection, primary antibodies used were: anti-mouse UCP1 (1:1000, mab6158, R&D Systems, Minneapolis, MN, USA), anti-mouse paxillin (1:200, 3127, Abcam, Cambridge, UK), anti-rabbit p38 (1:1000), anti-rabbit p-p38 (1:1000), anti-rabbit p-ATF2 (1:1000) (9212, 9211, 9225, Cell Signalling, Danvers, MA, USA), anti-mouse ATF2 (1:1000, WH0001386M2, Sigma-Aldrich) and anti-mouse β-actin (1:1000, A2228, Sigma-Aldrich) as a loading control. Bound primary antibody was detected with HRP goat-anti mouse or HRP goat-anti rabbit antibodies (P0447, P0448, Dako, Glostrup, Denmark). Chemiluminescence was captured with a ChemiDoc MP Imaging System (Bio-Rad). Quantification of the ATF2 phosphorylation was performed on the blots by normalizing the intensity of p-ATF2 and ATF2 to its corresponding β-actin and then dividing the normalized value of p-ATF2 by ATF2. The values are represented in bar graphs as a fold change to the iw condition. Intensity measurements were obtained using ImageJ software (NIH).