FIG. 3.

Interactions of CC3 with NPC, transportin, and exportin 4 are not sensitive to RanGTP. (A) FITC-labeled GST-M9 or GST-CC3 at 0.5 μM were added to permeabilized HeLa cells in transport buffer with or without 50 μg of HeLa cytosolic extract and 8 μM RanQ69L. After incubation for 20 min at room temperature, the cells were fixed and examined by fluorescence microscopy. (B) In vitro-translated transportin (lane 1) was incubated with GST-CC3 in the absence (lane 2) or in the presence of 1, 4, or 8 μM RanQ69L (lanes 3 to 5). Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by autoradiography. (C) His-exportin 4 at 5 μM was incubated with GST (lane 1) or GST-CC3 without RanQ69L (lane 2) or in the presence of 2.5, 5, or 10 μM RanQ69L (lanes 3 to 5) for 1 h at 4°C. Complexes were recovered with glutathione-Sepharose, resolved by SDS-PAGE, and detected by staining with Coomassie blue. (D) In vitro-translated transportin was incubated with His-RanQ69L in the absence (lane 3) or presence of 1, 4, and 8 μM GST-CC3 (lanes 4 to 6). Complexes were recovered with Ni-NTA agarose and analyzed by SDS-PAGE, followed by autoradiography. Lane 2 shows the amount of transportin that was incubated without RanQ69L and bound to the Ni-NTA agarose in a nonspecific manner. (E) In vitro-translated transportin was incubated with GST-M9 in absence (lane 2) or in presence of 1, 4, or 8 μM CC3 (lanes 3 to 5). Complexes were analyzed as described for panel B.