Amersham gel imager

Whole-protein lysates of liver tissue were extracted using 10 ml lysis buffer containing 9.8 ml NP40 cell lysis buffer (Invitrogen, Camarillo, CA, USA), 100µl protease inhibitor cocktail (Sigma-Aldrich), 100µl 50 mM β-Glycerophosphate (Invitrogen) and 33.3µl 0.3M phenyl-methylsulfonyl fluoride (Sigma-Aldrich). Bio-Rad DCTM Assay was used to measure protein concentration. We loaded 30µg of proteins onto an 8% to 12% sodium dodecyl sulfate-polyacrylamide gel, transblotted onto polyvinylidene difluoride membrane (Bio-Rad), blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20, and then incubated with the primary antibodies, including anti-PPARγ (1:1000, Abcam, ab209350) and anti-β-actin (1:2000, Merck, MAB1501). After incubating overnight, the membrane was then rinsed and incubated with anti-rabbit IgG, HRP-linked Antibody (1:2000, Cell Signaling, #7074s) or anti-mouse IgG, HRP-linked Antibody (1:5000, Cell Signaling, #7076s). The band complexes were recorded using Amersham Gel Imager (GE Healthcare, Life Science, USA) and quantified by a Fiji Image processing package. To ensure consistency, Western blot experiments were performed in duplicate. The results were presented as fold changes through divided by β-Actin and compared to the control.