FIG. 4.

Rpf1 is a nucleolar protein that requires Ran for nuclear import. (A) Wild-type yeast cells were cultured in liquid-rich media at 30°C and prepared for immunofluorescence microscopy. The samples were probed with affinity-purified rabbit antibodies against Rpf1 and with mouse monoclonal antibodies against the nucleolar marker protein Nop1 and then incubated with FITC-conjugated secondary antirabbit antibodies and with Texas Red-conjugated secondary anti-mouse antibodies and stained with DAPI. (B) Mutant prp20-1 cells bearing a temperature-sensitive mutation in the Ran-specific guanine exchange factor and containing a chromosomal RPF1-GFP allele were cultured at 25°C, shifted for 1 h to 37°C, and analyzed by immunofluorescence microscopy. The samples were probed with anti-GFP antibodies and FITC-labeled secondary antibodies to visualize Rpf1 and treated with DAPI to stain the DNA.