Figure 6. Effect of phosphorylation on AvrA function.

A Effect of phosphorylation on the ability of AvrA to inhibt S. Typhimurium-activated JNK activation. Henle-407 cells were infected for 30 min with a ΔavrA strain harboring a low copy plasmid expressing FLAG-epitope tagged AvrA or the indicated mutants (all expressed from the avrA native promoter), then chased in DMEM supplemented with gentamicin (100 µg/ml) for the indicated times. The activation of JNK and the phosphorylation of AvrA were monitored by western immunoblot analysis as indicated above. B Effect of AvrA phosphorylaton on its ability to inhibit S. Typhimurium-stimulated IL-8 transcription. HEK293 cells were co-transfected with pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection). Twenty four hours after transfection, cells were infected with S. Typhimurium strains expressing the indicated AvrA mutants and the stimulation of IL-8 transcription in infected cells was measured with the Dual-Luciferase Reporter Assay System (Promega) as indicated in Materials and Methods. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).