Figure 2

EPEC-induced tyrosine phosphorylation of cortactin depends on N-WASP. (A) Tyrosine phosphorylation of cortactin occurs in WT but not in N-WASP-deficient MEFs. WT, N-WASP-deficient and N-WASP reconstituted cells were infected with EPEC for three hours. The level of tyrosine phosphorylation of cortactin was assessed by Western blotting using an antibody against phospho-Y466 cortactin (upper panel). The same membrane was stripped and reprobed with anti-cortactin monoclonal antibody 4F11. N-WASP Western blotting was also performed to confirm the genotype of the MEFs. Actin was used as a loading control. Similar results were obtained in at least three independent experiments. (B) EPEC infection activates Src to similar extends in WT, N-WASP-deficient and R cells. WT, N-WASP-deficient and N-WASP reconstituted cells were infected with EPEC for three hours and analyzed for Src activation (phospho-Y416 Src) (upper panel). Medium and lower panels showed Src and actin blots, respectively. (C) Pervanadate treatment induces a robust phosphorylation of cortactin on tyrosine 466 in WT, N-WASP-deficient and R cells. Lysates of vehicle (DMSO)-treated MEFs and lysates of pervanadate-treated MEFs (diluted 1/500 *) were subjected to SDS-PAGE and Western blotted with antibody against phospho-Y466 cortactin. A second gel was loaded in parallel with the same amount of protein for both types of lysates and blotted for cortactin and actin for protein and loading controls, respectively (medium and lower panels). (D) EPEC infection induces Erk1/2 activation in WT but not N-WASP-deficient MEFs. WT, N-WASP-deficient and R cells were infected with EPEC for three hours. The level of Erk activation was assessed by Western blotting using a mAb specific for activated Erk phosphorylated on Thr202 and Tyr204 (upper panel). Medium and lower panels show Erk and actin blots for Erk protein and loading control respectively. Experiments were performed at least three times.