RavK-mediated cleavage of actin occurs during L. pneumophila infection.

A. Actin is cleaved by translocated RavK during bacterial infection. Cells expressing Flag-tagged actin were infected with relevant L. pneumophila strains for 2 h and cleared cell lysates was probed by immunoblotting with a Flag specific antibody. Tubulin was probed as a loading control. Similar results were obtained from five independent experiments and a representative blot was shown. B. The detection of RavK expression in L. pneumophila. The lysates of WT and the dotA- mutant were from exponentially grown bacteria whereas those of other strains were from bacteria grown at the post-exponential phase; the metabolic enzyme isocitrate dehydrogenase (ICDH) was probed as a loading control. C. RavK cannot be detected in saponin-soluble fractions of infected cells with RavK-specific antibody. SidC, a known Dot/Icm substrate was probed as a positive control for translocation and tubulin was probed as a loading control. D. Cleavage of endogenous actin. Lysates of cells similarly infected as described in C were probed for actin with tubulin as a loading control (left panel). E. The ratio of intensity between the bands representing actin and tubulin was quantified from three independent experiments. Note that a reduction in actin was not observed even in infections using the RavK overexpressing strain. N.S., not significant.