FIGURE 6.

Arsenite and eIF1 activity. A, 5′ AIRAP-GFP reporter was transfected alongside increasing amounts of the indicated eIF1 expression vector (25, 100, and 250 ng). 24 h post-transfection, cells were directly evaluated for GFP, eIF1, and PSMA1 content by immunoblot (IB). PSMA1 served as a loading control. B, SEAP reporter was introduced into 293 cells with eIF1 as indicated. SEAP levels were determined by immunoblot (left) or assayed kinetically for the reporter activity (right). * indicates a nonspecific band. C, non-transfected cells (lane 1) or transfected cells with the indicated SEAP reporter were untreated (lane 2) or arsenite-treated (lane 3) metabolically labeled, and immunoprecipitated against SEAP. * indicates a nonspecific band. Quantitation of three independent experiments is shown. Label incorporation decrease upon arsenite treatment in the kozak AUG reporter was set to 1, and relative increase in eIF1 AUG context repression is shown. D, presented is a surface structure of the human eIF1 (gray) and the rabbit 18 S RNA (yellow). The reported eIF1 residues contacting the 18 S (red) original positions in the yeast eIF1 are indicated in brackets. The phosphorylation site within eIF1 (Thr-72) is labeled in green. The figure is based on the published 4KZY PDB structural coordinates (33).