Figure 4From: Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cellsDetection 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. The negative control group cell lysate which was pre-incubated with GDP showed no band on the Western-blot membrane. The more intense bands found in the infected cells for anti-RhoA and anti-Rac1 compared to the uninfected cells indicated that more GTP-bound RhoA or Rac1 were precipitated from the infected cell lysate, which were activated upon T. gondii invasion.Back to article page