Figure 4.

Mutation of the NESs in full-length APC results in accumulation of protein in the nucleus. (A) Cos7 cells were transfected with plasmids that express Flag-tagged APC protein (APC) or Flag-APC with mutations in both NES1_APC and NES2_APC, APC(mNES1,2). At 24 h posttransfection, cells were prepared for immunofluorescence microscopy using an antibody raised against the Flag epitope (green, left panels). Nuclei were visualized using 4′,6-diamidino-2-phenlindole (DAPI) stain (blue, right panel). Scoring 100 cells for FLAG-tagged APC protein distribution revealed that mutation of both NES1_APC and NES2_APC resulted in accumulation of APC protein in the nucleus. (Scale bar, 10 μm.) (B) LMB treatment leads to an increase in endogenous APC protein in the nuclear fraction of HeLa cells. Following a 2-h treatment with LMB, HeLa cells were fractionated, and proteins from cytosolic and nuclear fractions were separated by SDS/PAGE. APC was identified by Western immunoblot using an ECL substrate. Bands from images captured with a Lumi-imager were quantitated, and values for five independent experiments were expressed as nuclear/cytoplasmic ratio (N/C) of full-length APC protein ±SD.