Figure 3: FAK and Src phosphorylate ACF7 at Y259.

(a) Immunofluorescence for ACF7 (green) and focal adhesions (vinculin, red). Note decreased localization of ACF7 in focal adhesions in PP2-treated cells or cells deficient for FAK. Boxed areas are magnified as insets. Scale bar, 20 μm. (b) In vitro kinase assays were performed on full-length ACF7 or ACF7-NT with recombinant FAK or Src proteins. Phosphorylation was analysed by SDS–PAGE and autoradiography. (c) Lysates are collected from cultured HEK293 cells expressing Myc-tagged ACF7-NT together with FAK and/or Src. Lysates were immunoprecipitated with α-Myc antibody. Immunoprecipitates (IP) and aliquots of whole-cell lysates (WCL) were analysed by SDS–PAGE and immunoblotting (IB) with different antibodies as indicated. (d) WT or FAK KO keratinocytes were transfected with plasmids encoding myc-tagged ACF7-NT and Src. After immunoprecipiation, proteins were subjected to SDS–PAGE and immunoblotting with different antibodies as indicated. (e) Lysates are collected from cultured cells expressing Myc-tagged ACF7-NT or ACF7-NT-Y259F mutant together with or without FAK and Src. Lysates were analysed by SDS–PAGE on immunoprecipiation and immunoblotting with different antibodies as indicated. The arrow denotes the band of ACF7-NT or NT-Y259F in lysates, and the ‘*’ denotes a non-specific background band present in the lysate. Note phosphorylation of WT ACF7 but not Y259F mutant leads to a band shift.