Fig. 1. TIAR accumulates in the cytoplasm of cells treated with actinomycin D. Cos cells grown on glass coverslips were incubated for 3 hours in normal medium (NT) or in medium containing cycloheximide (CHX; 20 µg/ml) or actinomycin D (ActD; 5 µg/ml) or both agents. Cells were then fixed, immunostained with anti-TIAR antibody and analyzed by confocal fluorescence microscopy as described in the Materials and Methods. Phase-contrast transmission images are also shown (Phase). Translational blockade by cycloheximide was verified by [³⁵S]methionine incorporation (data not shown).