Figure 7.
Expression and catalytic activity of an FAK/vinculin chimera. (A) A diagram of wild-type FAK and the FAK/vinculin chimera is shown. An initiation codon and epitope tag were fused in-frame with the FAK sequences beginning at codon 362. A termination codon was engineered immediately following codon 397 of the vinculin sequence. (B and C) CE cells transfected with empty RCAS (lane 1) or RCAS containing the sequences encoding wild-type FAK (lane 2) or the FAK/vinculin chimera (FV; lane 3) were lysed, and immunoprecipitations were performed using the BC2 polyclonal antiserum. One-half of each immunocomplex was analyzed by Western blotting using BC2. (C) The other one-half was incubated in an in vitro protein tyrosine kinase assay for 15 min in the presence of [γ-32P]ATP. Kinase reactions were terminated by adding an equal volume of 2× Laemmli sample buffer (Laemmli, 1970). The samples were analyzed by SDS-PAGE and autoradiography (B, exposure time of 1 h). The arrowheads indicate the position of the 97-kDa molecular weight marker.