Figure 5.

eEF1A is required for nuclear export of TD-NEM–containing proteins in living cells. (A–D) Silencing of eEF1A alters nuclear export of TD-NEM–containing proteins. MCF-7 cells were either mock-transfected or cotransfected with 100 nM eEF1A siRNA (2991 or 2804) or control siRNA and VHL-PK-GFP-NLS (A and B) or ΔC157-GFP (C and D) for 48 h before being submitted to the live cell FLIP nuclear export assay. In B cells were also treated with cycloheximide (CHX) for 1 h before cytoplasmic FLIP analysis. Cells were repetitively photobleached in a small cytoplasmic area (white box) within a cell of interest (dotted circle outlines the cell nucleus). The loss of nuclear fluorescence was monitored and plotted on a graph. The line graphs represent mean values obtained from the indicated number of cells used (n) per condition. Scale bars, 10 μm.