Phospho p ampkα

Mouse embryo fibroblast 3T3-L1 (ATCC^®CL173) and normal human primary subcutaneous preadipocytes (ATCC^® PCS210010) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified Eagle’s medium high glucose (DMEM), penicillin/streptomycin and L-glutamine were purchased from Mediatech, Inc. (Manassas, VA). PPARγ (Cat# 2443S), C/EBPα (Cat# 2295S), FAS (Cat# 4233S), HMGB2 (Cat# 14163S), AMPKα (Cat# 5832S) and phospho(p)-AMPKα (Cat# 50081S-Thr172), Akt1(Cat# 75692S) and p-Akt1 (Cat# 9018S-Ser473) antibodies were from Cell Signaling Technology (Boston, MA). GAPDH (Cat# G9545) antibodies were from Sigma-Aldrich (St. Louis, MO). All secondary antibodies (Cat# 305-035-045) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All other chemicals were obtained from Sigma-Aldrich unless otherwise stated.

Total cell extracts were prepared using ice-cold lysis buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Sigma-Aldrich). Proteins (20 μg/lane) were separated by SDS-polyacrylamide gel electrophoresis (10–15% gels) and transferred onto nitrocellulose membranes (Sigma-Aldrich). Membranes were blocked in 5% non-fat milk in TBS/0.1% Tween 20 for 2 h prior to immunoblotting overnight with antibodies against LC3B (1:500, ab48394; Abcam), phospho (p)-AMPKα (1:500; #2535, Cell Signaling Technology, Inc., Shanghai, China), p-mTOR (1:1000; #2971, Cell Signaling Technology), p-NF-κB (p65) (1:500; sc-166,748, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), TGFβ1 (1:1000; ab31013, Abcam), p-JAK2 (1:1000; ab195055, Abcam), p-STAT3 (1:800; ab30647, Abcam), Beclin-1(1:500; ab55878, Abcam) and β-actin (1:1000, Santa Cruz Biotechnologies). Incubation with the secondary fluorescent-labeled antibody (Alexa Fluor 488) was performed for 2 h at room temperature in the dark. The proteins were visualized by enhanced chemiluminescence (Amersham Bio-sciences, NJ, USA).