FIG 7.
Purified EspC directly cleaves immunoprecipitated FAK. HEp-2 cells were lysed, and protein extracts were incubated with primary antibody anti-FAK plus protein A-agarose overnight. Immunoprecipitated FAK was incubated with purified EspC or EspCS256I for 30 s or 1, 5, 10, or 15 min. Reactions were stopped with Laemmli buffer, and products were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and analyzed by immunoblotting using anti-FAK (full-length protein), anti-FAK-NH2, anti-FAK-COOH, and anti-EspC as primary antibodies and HRP-conjugated anti-isotype secondary antibody. The reaction was developed using Western blotting chemiluminescence reagent. HC, heavy chains of antibodies used for the immunoprecipitation (visualized as a loading control).