Nuclear import of FGFR1 is mediated by importin β. (a) Coimmunoprecipitation of importin β with FGFR1. Cells were untreated or subjected to ATP depletion by treatment with oligomycin B and 2-deoxyglucose for 2 h, in the absence or presence of FGF-2, and separated into cytosolic (C) and nuclear (N) fractions. FGFR1 was immunoprecipitated from equal amounts of protein, and complexes were separated by SDS-PAGE and immunoblotted for importin β. The whole cell lysate is shown for comparison, the immunoglobulin heavy chain (IgG) is indicated, and molecular weights are indicated in kD. (b–e) Nuclear translocation of FGFR1 in Swiss 3T3 fibroblasts was examined using an in vitro nuclear import assay (Adam et al. 1990), and cells were analyzed by FGFR1 immunostaining and confocal microscopy. Unpermeabilized cells (b) were untreated or incubated for 30 min with FGF-2. Digitonin-permeabilized cells (c–e) were incubated for 30 min with FGF-2 in the absence or presence of exogenous cytosol (c), with mock- or importin β–depleted exogenous cytosol (d) or with exogenous cytosol plus mouse IgG or a neutralizing antibody against importin β (e). Depletion of importin β from exogenous cytosol was confirmed by immunoblotting (d). Bar, 10 μm.