Evidence for the presence of a PbeIF2α phosphatase in salivary gland sporozoites. (A) Wild-type sporozoites were incubated at 37°C for 30 and 60 min, and then levels of PbeIF2α-P and total PbeIF2α were assayed by immunoblots. In B–E, wild-type and PbeIK2 (-) sporozoites were dissected from mosquito salivary glands in the presence (+) or absence (-) of 1 uM salubrinal (Sal) at room temperature, and then the medium was replaced by DME supplemented with 10% FBS. (B) Immunoblot assays for levels of PbeIF2α-P and total PbeIF2α of wild-type and PbeIK2 (-) sporozoites treated with or without Sal. In A and B, immunoblots were repeated at least four times and densitometry values are shown below bands. (C) Infectivity of Sal-treated sporozoites to HepG2 cells were evaluated by counting EEF numbers 48 h after infection. Data are shown as the means ± SD of three independent experiments. (D) Sal enhanced infectivity of wild-type sporozoites to mice. C57BL/6 mice (six mice per group) were intravenously injected with 5 × 10³ wild-type sporozoites treated with or without Sal. 42 h later, liver parasite burden was measured by real-time PCR. Data are shown as the means ± SD of two independent experiments. (E) Effect of Sal on the kinetic incorporation of ³⁵S-Met/Cys by wild-type and PbeIK2 (-) salivary gland sporozoites at 37°C. Data are shown as the means ± SD of two independent experiments.