Figure 4. Down-regulation of p85α suppresses IGF-1-mediated PI3K/Akt downstream signaling in pancreatic acinar cells.

(A–C) The effect of p85α siRNA on IGF-1-mediated PI3K/Akt downstream signaling in isolated pancreatic acinar cells was assessed by Western blotting and densitometric analysis. Acinar cells were transfected with either p85α or NTC siRNA 2 days before treatment with IGF-1 (10 nM) for 2 h. (A) Cellular protein extracts were subjected to Western blot analyses of p85α to confirm successful knockdown. Levels of p85α were normalized by the corresponding density of β-actin. (B) Cellular protein extracts were subjected to Western blot analyses of p-Akt, Akt, p-mTOR, mTOR, p-p70S6K, p70S6K, p-GSK3β, and GSK3β. (C) Densitometric analysis of (B). Levels of p-Akt, p-mTOR, p-p70S6K, and p-GSK30β, were normalized by the corresponding density of total Akt, mTOR, p70S6K and GSK3β respectively. Similar results were obtained from 3 independent experiments. Values are mean ± SD; n=3. *p<0.05 and ***p<0.001 comparing p85α siRNA with NTC siRNA treatment. †p<0.05 and ††p<0.01 comparing IGF-1 with control PBS treatment in pancreatic acinar cells transfected with NTC siRNA. (D) The effect of wortmannin on IGF-1-mediated PI3K/Akt downstream signaling in isolated pancreatic acinar cells was assessed by Western blotting. Isolated pancreatic acinar cells were treated with wortmannin (100 nM) 30 min prior to IGF-1 (10 nM) stimulation and harvested 120 min later. Cellular protein samples were subjected to Western blot analyses of p-mTOR, mTOR, p-p70S6K, p70S6K, p-GSK3β, and GSK3β. (E) Densitometric analysis of (D). Levels of p-Akt, p-mTOR, p-p70S6K, and p-GSK3β were normalized by the corresponding density of total Akt, mTOR, p70S6K, and GSK3β, respectively. Similar results were obtained from 2 additional independent experiments. Wort:wortmannin.