FIG 8.
Purified EspC directly cleaves immunoprecipitated paxillin. HEp-2 cells were lysed, and protein extracts were incubated with the primary antibody anti-paxillin plus protein A-agarose overnight. Immunoprecipitated paxillin was incubated with purified EspC or EspCS256I for 5, 10, and 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-paxillin as a primary antibody 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).