FAK- and adhesion-effects on v-Src substrate choice. (A) FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cells grown in adherent or suspension conditions as described in Experimental Procedures were analyzed by IB for levels of total Src, SrcpoY416 autophosphorylation or GAPDH (as a loading control). [Note that the decrease in Src protein, SrcpoY416 and GAPDH levels in lane 2 (second from left) is not reproducible; relative Src activation levels in adherent FAK-/-[v-Src] cells are typically comparable to those in adherent FAK+/+[v-Src] cells]. (B) Anti-phosphotyrosine (MAb4G10) IB from equal protein loads of FAK+/+[puro], FAK-/-[puro], FAK+/+[v-Src] and FAK-/-[v-Src] cell lysates. M, proteins markers in kDa. Decreased Src-induced tyrosine phosphorylation events in the absence of FAK are marked by arrows whereas increased tyrosine phosphorylation events in the absence of FAK are marked by asterisks. These data are typical of at least three independent experiments. A GAPDH IB is shown below as a loading control.