Fig. 3. SETA associates with FAK and PYK-2 as a dimer. To further analyze SETA's co-localization with focal adhesions, two major components of these structures, FAK (A) and PYK-2 (B), were immunoprecipitated from HEK293 cells that had been transiently transfected with SETA. Western blotting revealed that SETA could be recovered in both immunoprecipitates (A, lane 1; B, lanes 1,2), but was not recovered in control transfections (A, lanes 3,4; B, lanes 5,6). SETA appeared predominantly at about 160 kDa in the immunoprecipitates, while equal amounts of SETA at 85 kDa were present in lysates. Direct analysis (C) of SETA under strong (s; lanes 1,2) and weak (w; lane 3) denaturating PAGE conditions, as well as comparison with SETAcc (lane 4) lacking the coiled-coil domain, revealed that the 160 kDa band observed probably represents a SETA dimer mediated by homophilic interaction by this C-terminal domain, as shown previously (Borinstein et al., 2000; Watanabe et al., 2000). No SETA band at 160 kDa was found under strong denaturing conditions even when longer exposures (lane 1) were examined. This suggests that SETA preferentially associated with focal adhesion kinases as a dimer.