Figure 4: Phosphorylation of ACF7 at Y259 promotes F-actin binding.

(a,b) ACF7-NT, CH, plakin fragments or their mutants on Y259 were isolated from E. coli as His-tagged recombinant proteins. F-actin binding was examined by co-sedimentation assay. Pellet and supernatant, as well as an aliquot of pre-cleared protein sample (input) were immunoblotted with α-His6 to determine F-actin-binding affinity. Sup, supernatant. (c) Cells were transfected with plasmids encoding ACF7-NT or NT mutants together with or without FAK or Src. F-actin binding was examined by co-sedimentation assay. Pellet and supernatant,as well as an aliquot of pre-cleared cell lysate (input) were immunoblotted to assess the level of ACF7. (d) Cells were transfected with plasmid encoding full-length ACF7 or ACF7 mutants together with or without FAK or Src. F-actin binding was examined by co-sedimentation assay. Pellet and supernatant, as well as an aliquot of pre-cleared cell lysate (input) were immunoblotted to assess the level of ACF7. (e) Immunofluorescence for focal adhesions (vinculin, red), phosphor-ACF7 (green) and F-actin (blue) shows specific localization of phosphor-ACF7 at focal adhesions in WT but not ACF7 null cells. Boxed areas are magnified as insets. Scale bar, 20 μm. (f) Immunofluorescence for focal adhesions and phosphor-ACF7 in FAK null cells or WT keratinocytes treated with Src inhibitor, PP2. Scale bar, 20 μm.