Protein a g beads

Five micrograms of rabbit anti‐UCP1 antibody was first coupled to 30 μl protein A/G beads (Macgene) for 4 hr at 4°C on a rotating wheel in 250 μl IP buffer (20 mM Tris‐HCl, pH 8.0, 10 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.05% Triton X‐100, 0.05% NP‐40, 1 mM phenylmethylsulfonyl fluoride) with 1:100 protease inhibitor (Sigma) and 1:500 phosphatase inhibitor (Sigma). Meanwhile, 10 mg BAT at the transplantation region was lysed and ultra‐sonicated in 250 µl IP buffer and then precleaned with 30 µl protein A/G beads for 4 hr at 4°C. Then, protein A/G‐coupled UCP1 antibody was incubated overnight at 4°C with precleaned BAT lysate supernatant. Finally, after three washes (10 min each with 250 ul IP buffer), the beads with bound immune complexes were subjected to Western blotting SDS‐PAGE and silver staining, and the gel strip at UCP1 position (MW:33 kDa) was cut out and sent to Shanghai Bioprofile Inc. for UCP1 protein identification (rat UCP1 or mouse UCP1) through LC‐MS.

For Co-IP experiments, 5 μg of rabbit anti-KBTBD8 or rabbit anti-Cul3 antibody was first coupled to 30 μl protein-A/G beads (Macgene, Beijing, China) for 4 h at 4 °C on a rotating wheel in 250 μl IP buffer (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.05% Triton X-100, 0.05% NP-40, 1 mM phenylmethylsulfonyl fluoride) with 1:100 protease inhibitor (Sigma) and 1:500 phosphatase inhibitor (Sigma). Meanwhile, 600 oocytes were lysed and ultra-sonicated in 250 µl IP buffer and then pre-cleaned with 30 µl protein A/G beads for 4 h at 4 °C. Then, protein A/G-coupled KBTBD8 or Cul3 antibody was incubated overnight at 4 °C with pre-cleaned oocyte lysate supernatant. Finally, after three washes (10 min each with 250 ul IP buffer), the beads with bound immune complexes were subjected to western blotting with KBTBD8 or Cul3 antibody in parallel.

Five micrograms Rabbit anti‐Fam70a antibody, Rabbit anti‐Wnt5a, Rabbit anti‐beta‐catenin or Rabbit IgG was first coupled to 30 μL protein A/G beads (Macgene) for 4 hours at 4°C on a rotating wheel in 250 μL immunoprecipitate (IP) buffer (20 mmol/L Tris‐HCl, pH 8.0, 10 mmol/L EDTA, 1 mmol/L EGTA, 150 mmol/L NaCl, 0.05% Triton X‐100, 0.05% Nonidet P‐40, 1 mmol/L phenylmethylsulfonyl fluoride) with 1:100 protease inhibitor (Sigma) and 1:500 phosphatase inhibitor (Sigma). Meanwhile, 600 oocytes were lysed and ultrasonicated in 250 µL IP buffer and then pre‐cleaned with 30 µL protein A/G beads for 4 hours at 4°C. After that, protein A/G‐coupled Rabbit IgG or specific antibody was incubated overnight at 4°C with pre‐cleaned oocyte lysate supernatant. Finally, after being washed three times (10 minutes each with 250 µL IP buffer), the resulting beads with bound immunocomplexes were subjected to SDS‐PAGE and silver staining.