Figure 3.

Inhibitors of RNA PolII-dependent transcription block the interaction between VHL and eEF1A. (A) ActD and DRB hinder the interaction between eEF1A and VHL. MCF-7 cells transiently expressing F-GFP or F-VHL-GFP-NLS were untreated or treated with 8 μM ActD or 25 μg/ml DRB for 3 h before harvesting cells. Lysates were immunoprecipitated with anti-Flag beads and immunoblotted with Flag or eEF1A antibodies. (B and C) ActD inhibits TD-NEM–mediated nuclear export of VHL. MCF-7 cells transiently expressing VHL-PK-GFP-NLS were either untreated or treated with 2 μM ActD for 1 h before being submitted to cytoplasmic FLIP analysis. Cells were repeatedly bleached in a small cytoplasmic region (white squares) within cells (dotted circles outline the nuclei of bleached cells). The loss of nuclear fluorescence was monitored (C), calculated, and plotted on a graph (B). Scale bar, 10 μm.