FIG. 5.

Translocation of the AnkB variants into host cells. (A) Translocation of AnkB encoded by mutant alleles into U937 cells was determined at 1 h postinfection using adenylate cyclase assays and cyclic AMP (cAMP) levels as readout. Data points are the average cyclic AMP concentrations per well for a representative experiment performed three times in triplicate. Error bars represent standard deviations. (B) Equivalent expression of the AnkB-Cya fusion alleles in L. pneumophila was determined by immunoblot assays of Cya-AnkB fusions expressed in L. pneumophila. Proteins derived from equivalent numbers of bacteria (1 × 10⁸) were loaded onto an SDS-polyacrylamide gel, and Cya fusion proteins were detected on Western blots probed with an anti-M45 antibody, recognizing the N-terminal M45 epitope on all Cya fusions. The blots were reprobed with anti-CAT antibodies, which showed equivalent expression of another protein encoded on the same reporter plasmid.