Fastprep ribolyser

AT samples were homogenized with a FastPrep Ribolyser (MP Biomedicals, Elsene, Belgium) in 10 mM Na phosphate, pH 7.2, 150 mM NaCl, 1% Triton, 0.1% sodium dodecyl sulphate (SDS), 0.5% Na deoxycholate, 0.2% NaN₃, containing a protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Protein concentrations were determined with the BCA protein assay (Thermo Fisher Scientific, Rockford, IL, USA). An equal amount of protein was loaded in each well of a 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA). Gels were transferred onto a 0.45 μm nitrocellulose membrane and blocked in 5% nonfat dry milk (Bio-Rad) in 10 mM Tris–HCl buffer containing 150 mM NaCl and 0.05% Tween 20 at pH 7.6 (TBST) for 3 h. Subsequently, membranes were probed with the following primary antibodies: β-actin (Cell Signaling Technology; CST, Leiden, The Netherlands), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, CST), peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1α, Novus Biologicals, Abingdon, UK), and UCP-1 (Sigma-Aldrich, Darmstadt, Germany). Secondary antibodies were species-appropriate horseradish peroxidase-conjugated antibodies (Dako, Glostrup, Denmark, 1:2000) diluted in TBST containing 5% nonfat dry milk. Signals were detected with Enhanced Chemiluminescence (Thermo Fisher Scientific) (http://dx.doi.org/10.17504/protocols.io.kbfcsjn).

A Western blot for uncoupling protein-1 (UCP-1) was performed as described previously [7]. Briefly, BAT adipose tissue was homogenized using the FastPrep ribolyser (MP Biomedicals) in a buffer consisting of 10 mM Na phosphate pH 7.2, 150 mM NaCl, 1% Triton, 0.1% sodium dodecyl sulfate (SDS), 0.5% Na deoxycholate, 0.2% NaN₃ and a protease inhibitor cocktail (Thermo Fischer Scientific). The BCA protein assay (Pierce) was used to determine protein concentration. An equal amount of protein was then loaded onto a 10% SDS-PAGE. Gels were transferred onto a 0.45 µm nitrocellulose membrane and blocked in 5% non-fat dry milk (Bio-Rad) in 10 mM Tris-HCl buffer with 150 mM NaCl and 0.5% Tween 20 pH 7.6 for 3 h. Hereafter, the membrane was probed with a UCP-1 antibody (Sigma-Aldrich). For the loading control, β-actin was used. A horseradish peroxidase-conjugated secondary antibody in TBST containing 5% non-fat dry milk was then added. Signals were detected using enhanced chemiluminescence (Thermo Fischer Scientific). Analysis was performed with Image J (NIH).

Tissue explants harvested at 6 or 24 h were kept overnight in 200 µL of RNAlater reagent and then frozen at −80 °C prior to extraction of RNA. RNA stabilized tissue explants were transferred to Lysing Matrix A (MP Bio-medicals, Santa Ana, CA, USA) and homogenized with a FastPrep Ribolyser (MP Biomedicals). Lysed tissue supernatants were collected, and total RNA purified using RNeasy mini kit (Qiagen, Venlo, The Netherlands) following manufacturer’s instructions.