Apolipoprotein b

Proteins from male Syrian golden hamster liver samples was extracted with RIPA buffer [50mmol/l Tris-HCl (pH 7.4), 1% NP-40, 0.5% sodium deoxycholate, 150 mmol/l NaCl, 0.1% SDS, EDTA] containing protease and phosphatase inhibitors. Protein concentrations were measured using a BCA-100 Protein Quantitative Analysis Kit. After denaturation, protein samples were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Millipore). Nonspecific binding sites were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 for 1h and then incubated with primary antibodies against MTTP (Affinity), PTEN, β-Actin (Beyotime Biotechnology), Akt, Akt-phospho-Ser473 (Cell Signaling Technologies), Foxo1a, or Apolipoprotein B (Abcam) overnight at 4°C. After three washes in Tris-buffered saline containing 0.1% Tween 20 the membranes were incubated with horseradish peroxidase conjugated secondary antibodies (anti-mouse or anti-rabbit IgG) for 1h and visualized by ECL detection (Bio-Rad). Quantitation was performed by Image J software.