FIG. 4.
Localization of CesD2 and EspD. (A) Whole ICC172(pICC246) bacterial cells (WC) and culture supernatant (S) were probed with polyclonal anti-His antiserum, demonstrating that CesD2 is intracellular.(B) EPEC strains E2348/69, ICC172, and ICC172(pICC246) were fractionated into C, IM, OM, and P fractions. Samples were analyzed by Western blotting and probed with polyclonal anti-His antiserum (ICC172-pICC246) and monoclonal anti-EspD antibody (strains E2348/69, ICC172, and ICC172-pICC246). His6-CesD2 was detected mostly in the inner membrane, with small amount detected in the cytoplasm. EspD was detected in IM and OM fractions from the three strains. Samples were also probed with anti-β-galactosidase, anti-Etk, anti-intimin, and anti-MBP to verify fraction enrichment. Anti-β-galactosidase, anti-Etk, anti-intimin, and anti-MBP reacted with bands of the size expected for β-galactosidase (116 kDa), Etk (81.2 kDa), intimin (94 kDa), and MBP (43 kDa), respectively. As predicted, β-galactosidase and Etk were detected only in C and IM fractions, respectively. Intimin, which is an outer membrane protein, was also detected in the IM fraction, probably representing the unprocessed form of the molecule. MBP was detected in the periplasmic preparation, while it was also present in some of the C fractions. The upper band that reacted with the MBP antiserum is likely to represent a cross-reactivity with another cytosolic protein. Molecular mass markers are shown at right, and fractionation markers are identified on the left side of the panel.