Puromycin incorporation. HeLa cells were pre-treated with cycloheximide (100 µg/ml; 15 min) to slow ribosomes, pulsed with puromycin (puro; 91 µM; 0–60 s), fixed, puromycylated peptides immuno-labeled with Cy3, DNA stained with DAPI, and images collected.

(A-D) Typical confocal sections through the centre of nuclei. A 5-s pulse gives bright nuclear foci. After 30 s, cytoplasmic and nuclear signals are more similar and diffuse. After 60 s, the peri-nuclear region is the brightest. Bar: 10 µm. (E) Using wide-field images, Cy3 intensities (±SD; n = 20 cells) are expressed relative to the cytoplasmic value after a 60-s pulse.