FIG 4 .

An intact intravacuolar network is required for efficient ingestion of host protein. (A) Parasite strains were treated with LHVS during replication (or DMSO as vehicle control) in CHO cells transiently expressing cytosolic GFP. The results represent means ± SD from n = 3 independent determinations. Statistical analysis was by paired Student’s t test. (B) Schematic illustration for creation of the RHΔcplΔgra2 mutant. A PCR product carrying a phleomycin resistance cassette (BLE) flanked by GRA2 targeting sequences was transfected into RHΔcpl parasites for double crossover replacement of GRA2. (C) Primers indicated in panel A were used to verify the replacement of GRA2 with ble by PCR and agarose gel electrophoresis. (D) Cell lysates of RH and RHΔcplΔgra2 were immunoblotted with antibodies against CPL or GRA2 to confirm the absence of CPL and GRA2 expression in the RHΔcplΔgra2 mutant. Samples were immunoblotted also for actin as a loading control. (E) Ingestion is impaired in RHΔcplΔgra2 parasites. The values shown represent means ± SD from n = 3 independent experiments. Statistical significance by unpaired Student’s t test: *, P < 0.05; **, P < 0.01. ns, not significant (i.e., P > 0.05).