Sc517380

Proteins were extracted from iBAT using RIPA buffer (P0013B, Beyotime Biotechnology, Shanghai, China) with phosphatase and protease inhibitors (78442, Thermo Fisher Scientific, Waltham, MA, USA) and quantified in a BCA assay (23227, Thermo Fisher Scientific, Waltham, MA, USA). After extraction with 100% and 80% cold acetone, consecutively, proteins were resuspended in a loading solution (1610737, Bio-Rad, Hercules, CA, USA) with 2.5% (v/v) β-mercaptoethanol, heated at 95 °C for 3 min, separated by SDS-PAGE, and transferred onto PVDF membranes. Western blotting was conducted with antibodies against: phospho-p38 Mapk (1:1000, 4511T, Cell Signaling Technology, Danvers, MA, USA), p38 Mapk (1:1000, 8690T, Cell Signaling Technology, Danvers, MA, USA), Pgc1α (1:500, sc517380, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Ucp1 (1:500, U6382, Sigma, Burlington, MA, USA), and Gapdh (1:10000, MB001H, Bioworld, Nanjing, China). HRP-labeled secondary antibodies against rabbit (Bs13278, Bioworld, Nanjing, China) and mouse (20140714, Abgent, San Diego, CA, USA) Ig were used. Protein bands were visualized using chemiluminescent reagents (P10300, NCM Biotech, Suzhou, China) and scanned with an image analyzer (Amersham Imager 600, GE Healthcare, Chicago, IL, USA) followed by densitometric quantification.

Western blot analysis was performed as described in our previous report with some modifications [11 (link)]. Briefly, the cell proteins were extracted using RIPA lysis buffer containing a phosphatase inhibitor cocktail (Mei5 Biotechnology). Membranes were incubated overnight with primary antibodies at 4°C, including PGC-1α (1 : 1000; sc-517380; Santa Cruz Biotechnology, Dallas, TX, USA), LXR (1 : 1000; sc-377260; Santa), ApoE (1 : 800; sc-13521; Santa), β-actin (1 : 10000; sc-47778; Santa), ABCA1 (1 : 500; ab18180; Abcam, Cambridge, MA, USA), and ABCG1 (1 : 1000; ab52617; Abcam). Then, the blots were probed with horseradish peroxidase-conjugated secondary antibodies (1 : 10000; Mei5 Biotechnology), and the bands were visualized using an ECL detection kit. The images were obtained using the gel imaging system program (iBright CL1000; Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).