Fig. 3. Distribution of c-Myc in proteasome-inhibitor-treated cells. (A) c-Myc and c-Myc-GFP are nucleolar in proteasome-inhibitor-treated cells. COS-7 cells were cultured in the presence of 100 µM ALLN for 5 hours. Both c-Myc (upper panels) and c-Myc-GFP (lower panels) were localised to the nucleoli. The cells (lower panel) were transfected with 1 µg of c-Myc-GFP expression plasmid. Bar, 20 µm. (B) Time-lapse analysis shows redistribution of c-Myc-GFP in live cells after addition of 50 µM MG132. Confocal images were acquired every 120 seconds over a 3 hour period. The panel shows pictures selected at the times indicated. The arrows indicate accumulation of c-Myc-GFP in the vicinity of nucleoli. Nucleoli were identified by phase contrast microscopy as in A. Bar, 20 µm. (C) Proteasome inhibitor induced c-Myc redistribution is concurrent with its cellular accumulation. Whole cell extracts from COS-7 cells were analysed by SDS-PAGE and immunoblotting using anti-Myc C33 antibody. Lane 1 shows untreated cells. c-Myc protein levels increase after 3 and 6 hours of 100 µM ALLN treatment, lanes 2 and 3, respectively. ß-actin was used as an internal loading control.