Figure 6.

PI 3-kinase isoform expression in mesangial cells and the effect of wortmannin and LY 294002 on ATP-induced PKB phosphorylation. (A): RNA was prepared from quiescent mouse mesangial cells and reversed transcriptase-PCR of the p110α (α), p110δ (δ), p110γ (γ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as described in the Experimental Procedure Section. GAPDH runs at 470 bp, p110α at 779 bp, p110δ at 619 bp and p110γ at 621 bp. (B): Quiescent mesangial cells were pretreated with the indicated concentrations of wortmannin and LY 294002 for 20 min before stimulation for 5 min with 100 μM of ATP. Thereafter cell lysates, containing 70 μg of protein were subjected to SDS–PAGE (8% acrylamide gel) and Western blot analysis was performed using specific phospho-Ser⁴⁷³-PKB (upper panel), phospho-Thr³⁰⁸-PKB (middle panel) and total PKBα antibodies at a dilution of 1 : 1000, 1 : 1000 and 1 : 1600, respectively. Bands were visualized by the ECL method and evaluated on a densitometer. Data are representative of at least 3 independent experiments giving similar results.