The green fluorescence and the DIC images showing the invasion pr

The green fluorescence and the DIC images showing the invasion processes are provided in Additional file 1: Data S1 and Additional file 2: Data S2. The recruitment of Rho A and Rac1 GTPases into PVM is dependent on the GTPase activity We next investigated if intact GTPase activity was required for PVM recruitment. We used dominant negative mutants MG-132 concentration of Rho and Rac1 (RhoA-N19 and Rac1-N17 respectively) in our study. These mutants tagged with CFP were

overexpressed in COS-7 cells. At 48 hr post-transfection, the cells were infected with RH strain tachyzoites. Interestingly, the accumulation of these GTPases to the PVM was no longer seen when they were in these inactive forms, which constitutively bind only GDP (Figure 3). Thus, the recruitment of these Rho GTPases to the PVM only occurred when Rho GTPases retained normal activity. Figure 3 The recruitment of RhoA and Rac1 GTPases into parasitophorous vacuole membrane (PVM) is dependent on the GTPase activity (1000×). (A) The CFP-tagged dominant negative mutants RhoA N19 and Rac1 N17 were overexpressed in COS-7 cells and 48 hr post-transfection, the cells were infected with T. VX-770 in vitro gondii RH tachyzoites. All of these mutant

proteins did not accumulate on the PVM of T. gondii (arrowhead). (B) The CFP-tagged wild type RhoA and Rac1 were overexpressed in COS-7 cells and 48 hr post-transfection, the cells were infected with T. gondii RH tachyzoites. All of these wild-type proteins accumulated on the PVM of T. gondii (arrowhead). Bars: 10 μm. The Rho A and Rac1 GTPases were activated upon T. gondii tachyzoite invasion To determine if RhoA or Rac1 GTPases were actually Selleckchem Palbociclib activated following

T. gondii tachyzoite invasion, we used GST-tagged Rhotekin-RBD protein on agarose beads specific for RhoA or GST-tagged PAK-PBD protein bound agarose beads specific for Rac only to bind the GTP-bound RhoA or Rac1 in the cell lysate, but not very the GDP-bound form. Western-blot analyses detected increased amounts of GTP-bound RhoA and Rac1 from the infected cells compared with the uninfected cells (Figure 4), but no signals were detected in the negative control (16-HBE cells incubated with GDP) or the T. gondii infected groups. These results strongly suggest that T. gondii invasion results in the activation of RhoA and Rac1 GTPaes. Figure 4 Detection of RhoA and Rac1 activation in human 16HBE cells following T. gondii tachyzoites infection with Rho GST Pull-down assay. T. gondii RH tachyzoites infected human 16-HBE cells and uninfected cells were harvested and lysed. About 150 μg of the total protein from these two cell lysates was used in Rho pulldown assay. GST-tagged Rhotekin-RBD protein on agarose beads for RhoA or GST-tagged PAK-PBD protein bound agarose beads for Rac were used to bind and precipitate only the active form of RhoA or Rac1 in the cell lysate. In the Western-blot, actin was used as the equal protein loading control.

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