Nitrocellulose filter membranes

Proteins were isolated from ovine SVFs using a lysis buffer supplemented with protease inhibitors (Solarbio, Beijing, China), phosphatase inhibitors (Solarbio, Beijing, China), and phenylmethylsulfonyl fluoride (PMSF, Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose filter membranes (Solarbio, Beijing, China), and sealed in 5% skim milk for 1 h. Membranes were then incubated with primary antibodies (anti-β-actin: Immunoway, Beijing, China; anti-MEOX2: Proteintech, Wuhan, China; anti-adiponectin: Proteintech, Wuhan, China; anti-C/EBPα: Proteintech, Wuhan, China; anti-FABP4: Proteintech, Wuhan, China; anti-PPARγ: Proteintech, Wuhan, China) and secondary antibodies (LI-COR, Lincoln, NE, USA). After washing three times, the membranes were imaged using an Odyssey Clx imaging system (LI-COR, Lincoln, NE, USA).

To extract total proteins from cells, they were treated with a lysis buffer supplemented with protease inhibitors (Solarbio, Beijing, China, Cat. P6730), phosphatase inhibitors (Solarbio, Beijing, China, Cat. P1260) and phenylmethylsulfonyl fluoride (PMSF) (Solarbio, Beijing, China, Cat. P8340). The extracted proteins were loaded on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose filter membranes (Solarbio, Beijing, China, Cat. HATF00010). Then, the membrane was rinsed with PBS and sealed with 5% skim milk in PBS for 1 h. Western blotting was performed using the standard method for the following proteins with corresponding detection antibodies (in brackets): FNDC5 (1:1000, Abcam, Cambridge, UK, Cat. ab174833), anti-ERK1/2 and anti-phospho-ERK1/2 (1:2000, Cell Signaling Technology, Boston, MA, USA, Cat. 4370S). β-actin (1:2500, Proteintech, Wuhan, China, Cat. 205361AP) was used as an internal reference. After being washed with TBST, the membranes were incubated with a secondary antibody (1:10000, LI-COR, Lincoln, NE, USA, Cat. 92632211) for one hour at room temperature. Then, the membranes were washed with TBST again and imaged with the Odyssey CLX imaging system (LI-COR, USA).