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Figure 5


Fig. 5. RhoG is inactivated by YopE. (A) Cos-7 cells were co-transfected with myc-RhoG and either control plasmid (left lane), myc-TrioD1 (middle lane) or HA-Tiam1 (right lane). Active myc-RhoG was precipitated with GST-Elmo2NT and analysed by western blotting. Myc-RhoG and myc-TrioD1 were detected with anti-myc antibodies and HA-Tiam1 with anti-HA antibody. TrioD1 but not Tiam1 produced a strong RhoG activation. (B) Cos-7 cells were co-transfected with myc-RhoG and myc-TrioD1. After 24 hours, cells were infected with indicated Yersinia strains for 2 hours, lysed and subjected to GST-Elmo2NT pull-down. RhoG is downregulated by all strains except by {Delta}YopE strain. (C) Experiments were performed as in B but cells were infected with indicated bacterial strains. (D) In vitro GAP assay. GTP-hydrolysis was measured at 650 nm absorbance after co-incubation of indicated proteins for 20 minutes. YopE and YopE{Delta}MLD stimulated the GTPase activity of RhoG and Rac1 but not of Ras. p50RhoGAP was used as a positive control. Values are mean±s.d. (error bars) of three independent experiments.





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