The CFP-tagged Rho and Rac1 GTPases accumulated on the parasitophorous vacuole membrane (PVM) do not translocate toward epithelial growth factor (EGF) activation. Two paralleled groups of COS-7 cells were grown on coverslips and transfected with pECFP-RhoA and pECFP-Rac1 respectively. Forty-eight hr post-transfection, cells were starved overnight in serum-free DMEM. One group of cells was infected with T. gondii tachyzoites and the other group was kept uninfected. One hr post-infection, the infected cells were washed 3× with PBS to remove the unrecruited tachyzoites. Cells were site-activated with EGF for 5 min. (A) In uninfected cells, the CFP-tagged RhoA and Rac1 GTPases in the cytosol translocated to the host cell membrane (white arrowhead) in response to EGF activation. (B) In infected cells, the CFP-tagged RhoA and Rac1 were sequestered on the PVM without translocation toward the EGF, while the unassociated RhoA and Rac1 in the cytosol still translocated toward the EGF as in uninfected cells. More photographs provided in Additional file 4: Data S4 showing the RhoA and Rac1 sequestered on the PVM regardless the activation of EGF. Bar: 10 μm.