Figure 6.

eEF1A is required for nuclear export of TD-NEM–containing proteins in vitro. (A) MCF-7 lysate containing 0.5 mg of protein (see Figure 9D) was depleted of eEF1A or eEF2 using eEF1A or eEF2 specific antibodies (see Materials and Methods for details). The depletion of eEF1A and eEF2 was assessed by Western blot analysis. Actin was used as a control. (B and C) MCF-7 cells transiently expressing the indicated constructs were permeabilized with digitonin and incubated with transport buffer alone, MCF-7 lysate, or MCF-7 lysate that was depleted of eEF1A or eEF2. Where indicated cells were pretreated with 8 μM ActD, 25 μg/ml DRB, or 10 μM LMB for 3 h before permeabilization with digitonin, and the nuclear export assay was performed using undepleted MCF-7 lysate containing the same concentrations of drugs as was used in the pretreatment. All conditions were performed in the presence of ATP-, GTP-, and ATP-regenerating system. Nuclear export was monitored by loss of nuclear fluorescence (B). Relative loss in nuclear fluorescence was calculated and plotted on a graph (C).