Figure 4

Cortactin binding to Tir in N-WASP-deficient cells infected by EPEC. (A) Tir is abundant in membrane-enriched fractions. EPEC-infected and uninfected P100 monolayers of WT, N-WASP-deficient, and R MEFs were lysed in imidazole buffer, fractionated, subjected to SDS-PAGE and blotted with anti-Tir mAb. As previously described, Tir appears as a doublet whose upper band is enriched in the membrane fractions (pellets). The signal from the anti-actin antibody is shown as a loading control. (B) Cortactin binds Tir in cells through its N-terminal domain. Cell lysates of uninfected and EPEC- infected WT, N-WASP-deficient, and R (R) MEFs were analyzed in pull-down experiments using GST fusion proteins as follows: cortactin N-terminal truncation mutant (NH2), the isolated SH3 domain (SH3), and GST alone. Western blotting with anti-Tir monoclonal antibody revealed a unique band corresponding to the slower-moving band. This band was present only in the NH2 pull-down conducted on all three types of lysates. These experiments were performed at least three times.