Fig. 3. Effect of the chronic presence of insulin and gliclazide on phosphatidylinositol 3-kinase (PI 3-kinase) activity in C2C12 myotubes. C2C12 cells, differentiated in the absence (MF) or in the chronic presence (MFI) of insulin, were washed with Krebs-Ringer phosphate (KRP) buffer as described in Materials and methods, followed by stimulation with insulin (100 nmol l^-1) for 10 min. PI 3-kinase activity in anti-IRS-1 (insulin receptor substrate 1) immunoprecipitates was measured as described under Materials and methods. Cells were treated with 2 mmol l^-1 gliclazide during the last 24 h of differentiation. A representative autoradiogram for three independent experiments is shown (A). The relative density of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P₃] spots (B) was quantified by densitometry and expressed relative to control samples (unstimulated insulin samples). Error bars represent the S.E.M. of three independent experiments (^*P<0.05).