Cortactin tyrosine phosphorylation by Src at focal adhesions. (A) Enhanced cortactin tyrosine phosphorylation in cells overexpressing Src. Cortactin immunoprecipitated from HEK293 cells overexpressing GFP-tagged FAK, Src or both was blotted with an anti-phosphotyrosine antibody. (B) Incompetent cortactin tyrosine phosphorylation by mutating Src phosphorylation sites. Myc-tagged cortactin C-terminus (myc-CT) or mutated C-terminus (Y421/466/475/482F) [myc-CT(Y/F)] was co-expressed with GFP-tagged constitutively active Src (GFP-Src-CA) or inactive Src (GFP-Src-IN) in HEK293 cells and immunoprecipitated with anti-myc antibody (α-myc). The tyrosine phosphorylation of myc-tagged cortactin mutants was detected with anti-phosphotyrosine antibody (α-PY). (C) Activation of Src at focal adhesions during cell adhesion. Bar, 10 μm. 10, 30 and 120 min indicate the time elapsed after the suspension of HEK293 cells were plated onto fibronectin-coated dishes. Cells were stained with anti-FAK and anti-phosphotyrosine418 Src antibodies. Actin was stained with phalloidin. (D) and (E) Immunofluorescence staining of cortactin, Tyr421 phosphorylated cortactin and paxillin in cells overexpressing GFP-tagged constitutively activated Src or inactive Src. Bar, 50 μm. NIH3T3 cells expressing GFP-tagged constitutively activated Src (G-Src-CA) or inactive Src (G-Src-IN) were stained with anti-cortactin (Cttn), anti-paxillin (Pax) and anti-phosphotyrosine421 cortactin (pCttn) antibodies.