The more intense bands found in the infected cells for anti-RhoA and anti-Rac1 compared to the uninfected cells indicated Selleck Doramapimod that more GTP-bound RhoA or Rac1 were precipitated from the infected cell lysate, which were activated upon T. gondii invasion. The recruitment of RhoA to T. gondii PVM

is dependent on different RhoA domains In order to define what motifs are vital to the recruitment of Rho GTPases to the PVM, we concentrated on the study of Rho A as a representative protein. Sequential deletion of RhoA by 10 amino acids with site-directed mutation from the parental plasmid pECFP-RhoA-WT generated 19 RhoA mutants. The different find more CFP-tagged, truncated RhoA plasmids (M1-M19) were transfected into COS-7 cells grown on coverslips in 6-well plates and analyzed by immunofluorescence microscopy. M2 (RhoAΔ11–20),

M3 (RhoAΔ21–30), M4 (RhoAΔ31–40), M6 (RhoAΔ51–60), M17 (RhoAΔ161–170) could not be observed on the PVM (Figure 5), indicating the decisive motifs were potentially the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box. The other mutants were all similarly recruited to the PVM as in wild-type RhoA (Additional file 3: Data S3). These results show that the GAP (GTPase-activating protein) interaction site, the GEF (guanine nucleotide exchange factor) interaction Vorinostat site, the GDI (guanine nucleotide dissociation inhibitor) interaction site, the Rho kinase (ROCK) effector interaction Resminostat site, the PKN/PRK1 effector interaction site, the Switch I region, the Switch II region, the G3 box and the G4 box were not the decisive motifs for the recruitment of

RhoA to the PVM. Figure 5 The recruitment of RhoA to T. gondii PVM is dependent on different RhoA domains (1000×). COS-7 cells were transfected with 3 μg of pEGFP-N1-RhoA mutants’ plasmids M1-M19, respectively. Forty-eight hr post-transfection, the cells were infected with RH strain tachyzoites of T. gondii. M2 (RhoAΔ11–20), M3 (RhoAΔ21–30), M4 (RhoAΔ31–40), M7 (RhoAΔ61–70) and M17 (RhoAΔ161–170) were found not to accumulate on the PVM (white arrowhead and white labeling), indicating that the integrity of the features (F) as follows are essential for the recruitment of RhoA to the PVM: F1-GTP/Mg2+ binding site [chemical binding site], F-7:mDia effector interaction site, F-10:G1 box, F-11:G2 box, F-14:G5 box.