Figure 3.

Transportin 1 is the nuclear import receptor for YB-1. (a) Nuclear localization of GST-GFP-YB-1-C2 was examined in a semi-intact cell system with HeLa cytosol. Digitonin-permeabilized HeLa cells were incubated with GST-GFP-YB-1-C2, HeLa cytosolic fraction, and an ATP regeneration system. Localization of GST-GFP-YB-1-C2 was examined by fluorescence microscopy (left panels). Phase-contrast images are also shown (right panels). In some experiments, ATP regeneration system was omitted, and apyrase was included instead (middle row). (b) FLAG-Transportin 1 expression vector, pCMV-3xFLAG-TRN1 (lanes 2 and 4), or empty vector (lanes 1 and 3) was transfected into HeLa cells. Two days later, cell lysates were subjected to immunoprecipitation with anti-FLAG antibodies. Immunoprecipitates and input lysates were analyzed by immunoblotting with antibodies against YB-1 and FLAG. (c) Permeabilized HeLa cells were incubated with 2 μM GST-GFP-YB-1-C2 with 4 μM Ran-GDP and an ATP regeneration system in the absence (top row) or presence (bottom row) of 0.5 μM transportin 1. Localization of GST-GFP-YB-1-C2 was examined by fluorescence microscopy (left panels). Phase-contrast images are also shown (right panels).