P sirt 1

Liver and soleus muscle tissues were solubilized in RIPA lysis buffer (Pierce, Rockford, IL) using Fast Prep 24G system (MP Biosciences, Santa Ana, CA). After exposure, HepG2 cells were solubilized in RIPA lysis buffer. Protein content was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL) and SDS samples were prepared. Equal amount of protein (100 μg per lane) were electrophoretically separated on SDS-polyacrylamide gel, followed by blotting onto PVDF membrane. Following the transfer, membranes were blocked with TBST (10 mmol/l Tris-HCl pH 7.4, 150 mmol/l NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk (blocking buffer) and incubated with the primary antibodies (diluted in blocking buffer overnight at 4°C) against SIRT-1 (Cell Signaling, cat. #9475), p-SIRT-1 (Cell Signaling, cat. #2314), Akt (Cell Signaling, cat. #9272), p-Akt (Cell Signaling, cat. #9271), AMPKα (Cell Signaling, cat. #5831), p-AMPKα (Cell Signaling, cat. #2535), NQO1 (Santa Cruz, cat. #sc-16464), β-actin (Cell Signaling, cat. #4970), and β-tubulin (Cell Signaling, cat. #2146). Membranes were incubated with goat anti-rabbit immunoglobulin (IgG) secondary antibody (Santa Cruz, cat. #sc-2030) for 1 h at room temperature, and washed 5 times. Proteins were detected by using enhanced chemiluminescence. Semiquantitative analysis of Western blot images were performed using ImageJ.