RavK-mediated cleavage of actin occurs during <i>L</i>. <i>pneumophila</i> infection.

<p><b>A</b>. Actin is cleaved by translocated RavK during bacterial infection. Cells expressing Flag-tagged actin were infected with relevant <i>L</i>. <i>pneumophila</i> 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>B</b>. The detection of RavK expression in <i>L</i>. <i>pneumophila</i>. The lysates of WT and the <i>dotA</i><sup><i>-</i></sup> 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. <b>C</b>. 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. <b>D</b>. 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). <b>E</b>. 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.</p>