Colocalization of tyrosine phosphorylated cortactin and FAK at focal adhesions. Bar, 10 μm. (A) Dephosphorylation of Tyr421, 466 or 482-phosphorylated cortactin by alkaline phosphatase. Fixed COS7 cells were treated with 0.5 unit/μl calf intestinal alkaline phosphatase in 1 × TTBS (Tween/Tris-buffered saline, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween and 0.001% thimerosal) for 1 hour at room temperature, and then immunofluorescence-labeled with anti-FAK antibody (FAK), phalloidin (phalloidin), and with an anti-phosphotyrosine421, anti-phosphotyrosine466 or anti-phosphotyrosine482 cortactin antibody (pCttn421, pCttn466, pCttn482). Ctrl, without alkaline phosphatase treatment. (B) Colocalization of Tyr421-phosphorylated cortactin with FAK at focal adhesions. FAK, anti-FAK antibody staining. pFAK, anti-Tyr397-phosphorylated FAK antibody staining.