Fig. 7. Interference with cortactin expression or function inhibits invasion of S. aureus. (A) 293 cells were transfected with siRNA directed against either cortactin (Cort) or firefly luciferase (Luc) as a control. 24 hours later, cells were employed in gentamicin/lysostaphin protection assays with S. aureus. Values are means±s.d. of two independent experiments done in triplicate. Western blotting of whole cell lysates (WCL) with anti-cortactin antibody demonstrates gene silencing of cortactin (upper panel). The same membrane was probed with anti-actin antibodies as a loading control (lower panel). (B) 293 cells were transfected with the empty control vector (pcDNA) or cortactin mutants interfering with Arp2/3 association (W22A) or dynamin binding (W525K). Transfected cells were employed in gentamicin/lysostaphin protection assays with S. aureus. Values are means±s.d. of three independent experiments done in triplicate. (Lower panel) Western blotting of whole cell lysates (WCL) of the transfected cells with anti-FLAG antibody demonstrates expression of cortactin mutants. (C) 293 cells were transfected with the empty control vector (pcDNA), cortactin mutated at tyrosine residue 421 (Y421F), or mutated at three C-terminal tyrosine residues (Y421,466,482F). Cells were infected with S. aureus for 2 hours and the number of internalised bacteria was determined in gentamicin/lysostaphin protection assays. Values are means±s.d. of three independent experiments done in triplicate. Whole cell lysates (WCL) probed with anti-FLAG antibody demonstrate the expression of transfected cortactin constructs (lower panel).