Localization of CagI and CagL in bacterial cell fractions.

<p>(A) <i>H. pylori</i> cells grown in liquid culture for 24–48 hours were lysed by ultrasonication and subsequently fractionated into soluble and insoluble proteins by ultracentrifugation. Ultracentrifugation pellets containing membrane-associated and other insoluble proteins were extracted with 1% triton X-100 to separate outer membrane-associated (TX100 insol) from inner membrane-associated (TX100 sol) proteins. Comparable amounts of each fraction were analysed by immunoblot for their CagI and CagL content. As controls, immunoblots against the outer membrane-associated proteins CagX and AlpB, and the partly soluble and partly inner membrane-associated protein RecA were used. Representative immunoblots are shown. CagI and CagL bands are indicated by arrowheads. (B) Ultracentrifugation pellets were resuspended and subjected to isopycnic density gradient centrifugation on 25–60% sucrose gradients. Fractions were collected from the gradients and analyzed by immunoblotting with the indicated antisera. CagI and CagL bands are indicated by arrowheads. (C) Bacteria were subjected to limited proteolytic digestion by proteinase K. Equal amounts of untreated control cells (P12) and proteinase K-treated bacteria (P12 PK) were analyzed by immunoblot with the indicated antisera.