Fig. 4. The SETA binding partner AIP1 is associated with focal adhesion kinases. Analysis of the presence of the SETA-binding partners AIP1 and c-Cbl within focal adhesions revealed that AIP1 could be recovered by immunoprecipitation of FAK (A, lane 1) and PYK-2 (B, lanes 1,2) from transiently transfected HEK293 cells, whereas c-Cbl was exclusively and weakly associated with PYK-2 (B, lanes 3,4). Changing the cell density from low or non-confluent (l) to high or confluent (h) increased the amount of AIP1 associated with PYK-2, but did not influence the amount of c-Cbl. Neither protein had an influence on the concentration of PYK-2 under these conditions. Finally, the AIP1-binding partner ALG-2 was also found to co-precipitate with PYK-2 (C), indicating that an extended SETA-associated protein complex was intimately associated with focal adhesions. Please note that relative mobilities of proteins in Nu-PAGE gels is different from conventional Laemmli gels with stronger denaturation (see Materials and Methods).