FIG 4.
EspC cleaves fodrin (alpha II spectrin) within the 11th and 9th repetitive units. (A) Cleavage of epithelial fodrin by EPEC, EPEC ΔespC, EPEC ΔespC(pespC), or EPEC ΔespC(pespCS256I). HEp-2 cells were infected with the strains indicated above for 2 or 6 h. Infected cells were lysed, and the proteins were analyzed by immunoblotting using anti-fodrin antibody. The reaction was visualized using HRP-labeled rabbit anti-mouse antibodies and developed using Western blotting chemiluminescence reagent. The subproducts of fodrin degradation are indicated with asterisks, and the original fodrin is indicated with an arrow. Samples for all panels are identified at the top of each lane. (B) Cleavage of GST-fodrin by EspC. Purified GST-fodrin (2.5 μg) was incubated with EspC (1 μg) for 4 h and then separated by SDS-PAGE. Subproducts of GST-fodrin degradation by EspC containing the amino terminus (43 and 34 kDa) were sequenced. Sequencing of the 43-kDa subproduct showed that the first cleavage site is localized within the 11th repeat unit, between Q1219 and L1220, while the 34-kDa subproduct showed that the second cleavage site is localized within the 9th repeat unit, between D938 and L939.