Temporal regulation of SidH is mediated by ubiquitin ligase LubX and host proteasome.

<p>(A) Time course of intracellular SidH and LubX levels after infection with <i>L. pneumophila</i> strain producing the triple FLAG-tagged SidH. CHO-FcγRII cells were infected and digitonin extracts were prepared at indicated time after infection as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001216#ppat-1001216-g003" target="_blank">Figure 3B</a>. The Ni lane denotes the non-infected control. Immunoprecipitation and immunoblotting was carried out as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001216#ppat-1001216-g003" target="_blank">Figure 3B</a>. The asterisks denote a non-specific signal. (B) Shutdown of SidH requires ubiquitin ligase activity of LubX. CHO-FcγRII cells infected with <i>L. pneumophila</i> strains carrying wild-type <i>lubX</i> gene or <i>lubX</i>I39A U-box1 dead mutant were analyzed as in panel A. (C) Shutdown of SidH requires host proteasome. CHO-FcγRII cells were treated with 10 µM MG132 or 0.1% DMSO from 30 minutes before infection with <i>L. pneumophila</i>. (D) Shutdown of SidH requires bacterial protein synthesis after infection. <i>L. pneumophila</i> was pretreated with 100 µg/ml gentamicin (Gm) or water for 30 minutes and used for infection. (E) Delayed delivery of LubX results in proteasomal degradation of SidH at later stages of infection.</p>

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