Ab106410

The following antibodies were used for western blotting: rabbit monoclonal anti-CD24 (1:500, catalog number ab179821, Abcam, MA, USA), rabbit monoclonal anti-CD44 (1:500, catalog number ab189524, Abcam), rabbit recombinant multiclonal anti-CD133 (1:800, catalog number ab278053, Abcam), mouse monoclonal anti-Nanog (1:1000, catalog number ab173368, Abcam), rabbit monoclonal anti-OCT4 (1:500, catalog number ab200834, Abcam), mouse monoclonal anti-SOX2 (1:1000, catalog number ab79351, Abcam), rabbit polyclonal anti-DLG5 (1:1000, catalog number ab231283, Abcam), rabbit polyclonal anti-PRDM16 (1:1000, catalog number ab106410, Abcam), mouse monoclonal anti-ErbB-2 (1:500, catalog number sc-33684, Santa Cruz Biotechnology, CA, USA), and mouse monoclonal anti-β-actin (1:1000, catalog number A5441, Sigma-Aldrich, MO, USA). Other primary antibodies and secondary antibodies and experimental procedures for immunoblotting are provided in the supplementary experimental procedures^{10 (link)}.

Firstly, paraffin embedded sections were deparaffinized and rehydrated, then washed with PBS and incubated with 0.1% Triton X-100 for 10 min, and then repaired the antigen in sodium citrate (pH 6.0) buffer by boiling 10 min in microwave oven. After cooling to the room temperature, sections were blocked in PBS containing 10% normal goat serum for 30 min at room temperature. Primary antibodies including PRDM16 (1:1.000, ab106410, Abcam) and PGC-1α (1:1,000, ab54481, Abcam) were then diluted in PBS and added to sections overnight at 4 °C. After washing in PBS, slides were then incubated with secondary antibodies that diluted in PBS containing 10% normal goat serum for 1 h at 37 °C. Slides were then mounted with Prolong Anti-Fade mounting medium containing DAPI (1:1,000, 28718–90-3, Sigma). All images were obtained using Leica SP5 scanning confocal microscope (Live cell Imaging Facility).

Adipose tissues were fixed in 4% PFA for 24 h at 4 °C, embedded in paraffin or tissue freezing medium (Leica) and sectioned to 8 μm. HE staining was conducted according to standard protocols. For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 h at room temperature, then incubated overnight at 4 °C with primary antibodies to mouse Foxp1 (Millipore, ABE68, 1:100), Ucp1 (Abcam, ab10893, 1:50), Prdm16 (Abcam, ab106410, 1:50). Subsequently, sections were incubated with secondary fluorescent-conjugated or HRP-conjugated antibodies at room temperature for 2 h in the dark. Samples were imaged by the Leica TCS SP5 confocal microscope, Leica DM2500, or Leica 3000B microscope. Transmission electron microscopy (TEM) of white and brown adipose tissue was carried out in accordance with a previous study.

Samples prepared with Laemmli sample buffer and were separated by 10% SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked with 10% skim milk in PBST and incubated at 4 °C overnight with rabbit anti-4-HNE antibody (1:1000; cat. no. PAB1295, Abnova) and then with secondary antibodies with HRP(1:10000; cat. no. GTX26721, GeneTex). Immunoblots for Ucp1, Prdm16, Pgc1α, Dio2, Cidea, Hsp70, and Gapdh were performed using rabbit polyclonal anti-Ucp1 antibody (1:1000, cat. no. GTX10983, GeneTex), rabbit polyclonal anti-Prdm16 (1:1000, cat. no. ab106410, Abcam), rabbit polyclonal anti-Pgc1α antibody (1:1000, cat. no. NBP1-04676PCP, Novus), rabbit polyclonal anti-Dio2 antibody (1:1000, cat. no. GTX81072, GeneTex), rabbit polycloncal anti-Cidea (1:1000, cat. no. ab8402, Abcam), rabbit monoclonocal anti-Hsp70antibody (1:4000. cat. no.ab45133, Abcam), and rabbit polyclononal anti-Gapdh antibody (1: 5000,GTX100118, GeneTex).

The frozen mouse tissues were homogenized in tissue protein extraction reagent (Thermo 78510) supplemented with a protease and phosphatase inhibitor mini tablet (Thermo 88668) per 10 mL and then centrifuged for 20 min at 12,000 ×g to remove cell debris. The total protein concentrations were determined using a BCA kit (Thermo 23225). The proteins were separated on a 7.5%–12.5% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck-Millipore). The membranes were blocked for one hour in TBST containing 5% skim milk and subsequently probed with primary antibodies overnight at 4 °C. Antibodies against β-TUBULIN (CST, 2146), RNF20 (Proteintech, 21625-1-AP), H2Bub (CST, 5546), H3K4me3 (abcam, ab8580), ATGL (CST, 2138), HSL (CST, 4107), pHSL (Ser660) (CST, 4126), pHSL (Ser565) (CST, 4137), pHSL (Ser563) (CST, 4139), PPARγ (CST, 2443), CEBPα (abcam, ab40764), UCP1 (abcam, ab10983), PRDM16 (abcam, ab106410), PGC1α (abcam, ab54481), Phospho-CEBPβ (Thr235) (CST, 3084), CEBPβ (Santa cruz, sc-7962), CYCLIND1 (CST, 55506S) and PCNA (CST, 2586) were used for western blot analysis. The blots were developed using anti-rabbit IgG, HRP-linked secondary antibody (CST, 7074) or anti-mouse IgG, HRP-linked secondary antibody (CST, 7076) and measured with an ECL-Plus system.