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. 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.
Na et al. BMC Microbiology 2013 13:125 doi:10.1186/1471-2180-13-125