Anti srebp 1c

Tissues and cultured cells were lysed with RIPA buffer supplied with protease inhibitor cocktail (Roche, Mannheim, Germany). Concentrations of protein were detected by the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Total protein extract (20 μg) was separated by 8% or 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The primary antibodies were as follows: anti‐AMPK, anti‐p‐AMPK, anti‐ACC, anti‐p‐ACC and anti‐SREBP‐1c (1:1000; Cell Signalling Technology, Beverly, MA). Immunoreactive bands were quantitatively analysed with ImageJ software.

Protein expression was evaluated by western blot. Frozen mouse epididymal fat tissue was homogenized in liquid nitrogen. Tissue was lysed in RIPA lysis buffer (Sigma) containing 1% PI. Cell lysate was kept on ice for 1 h and subsequently centrifuged at 13,000 rpm for 20 min at 4°C. Total proteins (30 μg protein/test) were isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (DC, Invitrogen). The membrane was blocked with 5% skim milk and subsequently incubated at 4°C with the following primary antibodies (1:5000 dilution): anti-AMPK, anti-SREBP-1c, anti-FAS, anti-ACC, anti-PPARγ, anti-C/EBPα, anti-FABP4, and anti-adiponectin (Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated with IgG HRP-conjugated secondary antibody (1:2000) for 2 h at room temperature. Ponceau S was used for staining the protein bands. β-actin was used as the loading control. Proteins were visualized using enhanced-chemiluminescence (ECL) location reagent and quantified with the ImageJ program.