Figure 3.

Effect of insulin on tyrosine phosphorylation of FAK, paxillin, and Src in CHO cells. (A) Confluent CHO-WT and CHO-IR cells were serum starved for 3 h and treated without or with insulin (100 nM) for times indicated to coincide with a total serum starvation time of 3 h. Cell lysates were prepared and immunoprecipitated (IP) with anti-FAK (α-FAK) and immunoblotted (IB) with α-FAK. Alternatively, cell lysates were immunoprecipitated with anti-PY antibody (α-PY) and immunoblotted with anti-FAK (α-FAK), anti-paxillin (α-PAX), or anti-Src (α-Src), as outlined in MATERIALS AND METHODS. Shown is one experiment representative of four. (B) The ECL-developed films were quantitated by scanning densitometry, and the optical absorbance was plotted in arbitrary units relative to untreated samples, which were ascribed a value of 1.0. (C) Cell lysates were immunoprecipitated with α-FAK, α-PAX, or α-Src and immunoblotted with α-PY, as outlined in MATERIALS AND METHODS. Shown is one experiment representative of two. In this experiment, the densitometric readings of the bands, in arbitrary units relative to unstimulated cells, were as follows: FAK IP: 1.0, 1.7, 1.4, 2.0 for CHO-WT cells and 1.0, 0.8, 0.09, 0.3 for CHO-IR cells; paxillin IP: 1.0, 1.2, 1.6, 1.5 for CHO-WT cells and 1.0, 0.7, 0.09, 0.6 for CHO-IR cells.