Fig. 2. Analysis of FA protein phosphotyrosine levels. Biochemical analysis of FA protein phosphotyrosine levels in cells expressing wild-type human 1 (HA1) or the swapped integrin chimers (SW1 or SßX). Cells were kept in suspension for 1 hour and allowed to spread on collagen IV (CN) or fibronectin (FN) for 1 hour, and phosphotyrosine levels were compared with the suspension control (S). (A) Phosphorylation of FAK at Y397 and Y576 were detected with site-specific pY antibodies (Biosource). Lysates were used neat (50 µg) for pY397 blots or immunoprecipitated (IP; 500 µg) with anti-FAK mAb (Upstate) for pY576 blots. Membranes were reprobed with anti-FAK (Transduction) or anti-talin (Sigma) mAbs. (B) Total phosphotyrosine levels in immunoprecipitates of paxillin or Cas. Both SW1 and SßX cells mediate normal FAK phosphorylation but are defective in Cas phosphorylation. SßX cells mediate wild-type levels of paxillin phosphorylation.