Sc 81433

After treatment, HepG2 cells were washed with ice-cold PBS and cellular protein was collected by scraping the cells into 50 μL protein extraction buffer, after which western blotting was carried out as described [27 (link)]. Membranes were probed with the antibody against SREBP-1c (sc-365,513), protein kinase B (Akt, sc-5298), phosphorylated protein kinase B (p-Akt^s473, sc-81,433), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-47,724) (all purchased from Santa Cruz Biotechnology), followed by incubation with horseradish peroxidase-conjugated second antibody. Immunoreactive bands were visualized by the enhanced chemiluminescence detection system.

The extracted cell total protein was loaded, separated by 10% SDS-PAGE, and the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with TBST solution containing 5% nonfat milk at room temperature for 1 h. The membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-human monoclonal PCNA antibody (ab92552; 1:1000 dilution; Abcam, UK), rabbit anti-human monoclonal cleaced casepase-3 antibody (ab2302; 1:1000 dilution; Abcam, UK), mouse anti-human monoclonal antibody to phosphorylated protein kinase B (p-Akt; sc-81433; 1:1000 dilution; Santa Cruz Biotechnology, USA), mouse anti-human monoclonal Akt antibody (sc-56878; 1:1000 dilution; Santa Cruz Biotechnology, USA), and rabbit anti-human monoclonal β-actin antibody (ab179467; 1:1000 dilution; Santa Cruz Biotechnology, USA).The membranes was washed by TTBS for 5 times, and then followed by HRP-linked secondary antibodies (ab6721 and ab6789; 1:1000 dilution; Abcam, USA) for 2 h at room temperature, and the protein signals were detected using an enhanced chemiluminescence reagent (Bio-Rad Laboratories, USA).