Figure 4.

Effect of APPL1_Asn94 Transfection on Akt-S⁴⁷³ and GSK3β-S⁸ Phosphorylation

HepG2 cells were transiently transfected with APPL1, APPL1_Asn94, or empty vector. After 48 hr, cells were stimulated with 100 nmol/l insulin for 5 min and then lysed. Phospho-AKT-S⁴⁷³ (A) or phospho-GSK3β-S⁸ (B) were evaluated by immunoblot analyses. In brief, equal amount of protein from cell lysates were separated by SDS-PAGE and probed with anti-phospho-AKT-S⁴⁷³ (A, upper blot), anti-AKT (A, middle blot), anti-APPL1 (A and B, lower blot), or anti-phospho-GSK3β-S⁸ (B, upper blot) and anti-GSK3β (B, middle blot) specific antibodies, respectively. Gel images were acquired with Molecular Imager ChemiDoc XRS (Biorad) and analyzed with Kodak Molecular Imaging Software 4.0 or IMAGEJ 1.40 g (Wayne Rasband, NIH). A representative blot for each condition is shown. Bars represent the percentage of AKT-S⁴⁷³ phosphorylation/AKT OD ratio (A) or GSK3β-S⁸/GSK3β OD ratio (B) in insulin-stimulated control cells. Data are means ± SD of three experiments in separate times.