FIG. 8.

Phosphorylation of eIF3a in wild-type and sty1 mutant cells. Wild-type (EA128) or sty1 mutant (DN7) cells expressing eIF3a-HA were harvested before treatment or after treatment with stress agents as indicated. Nondenaturing protein lysates were prepared as for Fig. 1 to 3, and HA-tagged eIF3a was purified with HA probe. Approximately equal amounts of total eIF3a were loaded in each lane. The position of a protein size marker is shown to the right. The eIF3a band is indicated; the band visible just above it in the SYPRO staining corresponds to eIF3c copurifying under native conditions, which migrates slightly slower than eIF3a (46). Upper panel, phosphorylated eIF3a detected with ProQ Diamond phosphoprotein stain. Lower panel, total eIF3a detected with SYPRO Ruby protein stain.