Phosphorylation of Eukaryotic Initiation Factor-2α during Stress and Encystation in Entamoeba Species
Fig 6
Polyribosome abundance in transgenic cell lines after 72 h of induction of protein synthesis.
RNA was isolated from the four transgenic cell lines after incubation in 5 μg mL-1 tetracycline for 72 h. The cell lines were the control cell line expressing luciferase, 209-Luc (A), the cell line overexpressing EheIF2α (B), the cell expressing the non-phosphorylable form of EheIF2α (C), and the cell line expressing the phosphomimetic form of EheIF2α (D). The RNA was resolved by sucrose gradient (15–45%) ultracentrifugation, which separates free ribosomes and monosomes (light fractions) from polysomes (dense fractions). The gradients were fractionated and the fractions were analyzed by UV spectrometry (254 nm). Representative profiles of at least three separate trials are shown. The percent of total absorbance in the dense polyribosome fractions (mean ± standard error, n≥ 3) is show in each panel. There is a statistically significant reduction in polyribosome abundance in cells expressing EheIF2α-S59D (*P<0.05) when compared to control cells (209-Luc).