Figure 2.

TD-NEM of VHL is required for interaction with eEF1A. (A and B) The transcription-dependent nuclear export sequence of VHL, encoded within residues 115–130, is required to interact with eEF1A and mediate nuclear export. (A) Cellular lysates from cells transiently expressing Flag-tagged VHL-GFP, Δ157-GFP, or Δ115–130-GFP were immunoprecipitated with anti-Flag beads and immunoblotted with anti-Flag and anti-eEF1A antibodies. Lanes referred to as “Beads” indicate that the Flag-beads were incubated with lysis buffer alone. (B) MCF-7 cells transiently expressing PK-GFP-NLS–tagged VHL or Δ115–130 were submitted to the live cell FLIP nuclear export assay. A small cytoplasmic region of the cell was photobleached repetitively and the loss of nuclear fluorescence, which is indicative of nuclear export activity, was monitored over time. The graph represents the relative loss of nuclear fluorescence over time. (C and D) Mapping the eEF1A-binding region within VHL. (C) Cellular lysates from cells transiently expressing the indicated constructs were immunoprecipitated and immunoblotted the same as in A. (D) Schematic diagram indicates deletion and truncation mutants of VHL that were submitted to immunoprecipitation with anti-Flag beads and immunoblotted with anti-Flag or anti-eEF1A antibodies. These mutants of VHL were also fused to PK-GFP-NLS and submitted to the live cell FLIP nuclear export assay. (+), (+ −), (− +), and (−) indicate, in a decreasing order, the ability of the fusion proteins to interact with eEF1A or export from the nucleus. ENP, experiment was not performed.