Rabbit anti β tubulin

Tissue lysate was obtained by the total protein extraction kit (Solarbio, Beijing, China). After protein samples transferring to PVDF membranes, we cut off the extra PVDF membrane according to the molecular weight of the target protein. Then, the remaining PVDF membrane was incubated with a primary antibody. Antibodies were diluted 1:500 for rabbit anti-UCP1 (Cat: 72,298; Cell Signaling Technology, MA, USA), 1:500 for rabbit anti-PGC1α (Cat: A12348; ABclonal, Wuhan, China), 1:1000 for rabbit anti-β-Tubulin (Cat: AC008; Abclonal, Wuhan, China), and 1:1000 for HPR-labeled goat anti-rabbit IgG (Beyotime, Shanghai, China). Finally, a ChemiDoc Imaging Systems (Bio-Rad, CA, USA) was used to detect immunoreactive proteins.

The proteins used in the experiments were extracted using the protein extraction kit (Bestbio, Shanghai, China). The following primary antibodies were used: dilution 1:1000 for rabbit anti-UCP1 (Cell Signaling Technologies, MA, USA), and dilution 1:3000 for rabbit anti-β-Tubulin (Abclonal, Wuhan, China). The primary antibodies were incubated at 4 °C overnight. HRP-labeled goat anti-rabbit secondary antibody was diluted at 1:1000 for 1.5 h at 37 °C. Finally, the proteins were detected by the ECL detection system (Beyotime, Shanghai, China).