Fig. 2. Effect of the chronic presence of insulin and gliclazide on tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in C2C12 myotubes. Samples were treated as described in Fig. 1. Cell lysate was immunoprecipitated (IP) with antibodies against IRS-1 and western immunoblotted (IB) with anti-phosphotyrosine (pTyr) antibody (A). The blots were stripped and reprobed with IRS-1 (B). Experiments were repeated three times and representative blots are shown. Phosphorylation levels of IRS-1 (C) were quantified by densitometry and expressed relative to MF (controls, differentiated in the absence of insulin) samples. Error bars represent the S.E.M. of three independent experiments (^**P<0.01). MFI, cells differentiated in the chronic presence of insulin.