Figure 4.

SG dissolution by emetine is due to polysome stabilization and occurs downstream of eIF-2α phosphorylation. (A) DU-145 cells were cultured in media alone (lane C), 0.5 mM arsenite for 30 min (A30), 0.5 mM arsenite for 90 min (A90), 0.5 mM arsenite for 90 min with addition of 10 μg/ml emetine after 30 min (A+E), 10 μg/ml emetine for 60 min (E), or 20 μg/ml puromycin for 60 min (P). Whole cell lysates were then separated by SDS-PAGE, transferred to nitrocellulose, and probed using antibodies specific for eIF-2α (bottom) or phosphorylated eIF-2α (top, p-eIF-2α). (B) Polysome profiles of control and treated cells. DU-145 cells were untreated (black open squares), or treated as described in A and subjected to sucrose gradient analysis as described in Results. A30, blue diamonds; A90 treatment, red squares; A+E treatment, green squares; E alone, blue squares. Polysome profiles were obtained by measuring the absorbance at 260 nm of individual fractions. Monosomes are found in fractions 2–5, and polysomes are found in fractions 7–12 (the top of the gradient is fraction 1). (C) Western blots of individual fractions. Fractions obtained from the gradients were subjected to PAGE in SDS and immunoblotting for PABP-I, TIA-1, and ribosomal subunit (Rib) proteins S3 and S19 as indicated. The lightest fractions are at the left, and the 80S and polysomal regions are indicated. 1, control; 2, arsenite treatment for 30 min; 3, arsenite treatment for 90 min; and 4, arsenite treatment for 90 min with emetine added during the last 60 min.