Fig 4.

eIF4E1^mAID-HA integrity is required for efficient tachyzoite replication and translation. (A) Analysis of eIF4E1^mAID-HA depletion over time upon addition of 500 µM IAA by western blot. The blot was probed with α-SAG1 as a loading control. Molecular weights are indicated in kDa. (B) Plaque assay of vehicle (0.5% dimethyl sulfoxide [DMSO])-treated parasites, parasites pulsed for 24 h with 500 µM IAA, or parasites treated with IAA for 6 days. Mean plaque numbers ± standard deviation were tested for statistical significance by a one-way ANOVA followed by a Student’s t-test assuming unequal variances. *P ≤ 0.01. (C) Analysis of parasite replication after a 16-h treatment with 500 µM IAA. The mean number of parasites per vacuole with standard deviation is shown. Statistical significance of the change between the mean number of parasites per vacuole was determined by Student’s t-test assuming unequal variances. *P ≤ 0.01. (D) Representative immunofluorescence assay images of eIF4E1^mAID-HA parasites treated with either DMSO vehicle or with IAA for 4 h. A 10-min pulse with 10 µg/mL puromycin to label newly synthesized proteins was included. eIF4E1^mAID-HA and puromycinylated peptides were visualized by immunofluorescence microscopy and DNA by DAPI staining. The translation elongation inhibitor CHX was added concurrently with puromycin to completely block protein synthesis as a control. Microscopy scale bar = 10 µm.