Fig. 5. Recruitment and phosphorylation of FAK accompanies S. aureus invasion. (A) FAK-deficient fibroblasts were transfected with GFP-FAK. Transfected cells were infected with rhodamine-labelled S. aureus at MOI 20 for 1 hour. The arrowheads point to accumulation of GFP-FAK in the vicinity of cell-attached S. aureus. Local recruitment of GFP-FAK (green line) to cell-associated bacteria (red line) was quantified by plotting the fluorescence intensity as detected in the GFP or rhodamine channels, respectively, against the distance. Bar, 10 µm. (B) FAK re-expressing fibroblasts were plated on poly-L-lysine, and either kept uninfected or infected at MOI 50 for 1 hour with S. aureus or S. carnosus, respectively. Samples were treated with pervanadate 10 minutes prior to lysis, and whole cell lysates (WCL) were analysed by western blotting with a monoclonal anti-phosphotyrosine (P-tyr) antibody (upper panel). Membranes were stripped and reprobed with anti-HA antibody (middle panel) or anti-cortactin antibody (lower panel). (C) FAK re-expressing cells were treated as in B and infected for the indicated times. WCLs were analysed by western blotting with anti-P-tyr antibody (upper panel) or anti-desmin antibody (middle panel). The same samples were immunoprecipitated (IP) with anti-cortactin antibodies and precipitates were analysed with anti-P-tyr antibody (lower panel).