Xenopus Cdc14α/β localize to the centrosome and nucleolus. A. Characterization of anti-XCdc14 antibodies. Affinity purified antibodies raised against the N- (aa 46–336) and C-termini (aa 376–577) of XCdc14α were used to Western blot Xenopus egg lysate. Arrows indicate a protein band that cross-reacts with both antibodies and that is competed away by pre-incubation of the antibodies with the immunizing antigen. The asterisk indicates a cross-reacting protein with X46-336 antibodies that partially competed by preincubation. This is likely to represent XCdc14β. B. XCdc14α/β localizes to the centrosome and nucleolus. Antibodies raised against the N-terminus of XCdc14α were used to immuno-stain Xenopus XTC cells. The cells were co-stained with the centrosomal marker γ-tubulin. The arrow in the upper left panel indicates XCdc14α/β localization to the centrosome in an interphase cell. XCdc14α/β localization to interphase nucleoli is also very prominent. C. XCdc14α is imported into nuclei formed in Xenopus embryonic extracts. Interphase Xenopus extracts were incubated with demembranated sperm (1000 sperm/μl) in order to induce nuclear formation. The time course was started by moving the reaction tubes to 23°C, and at the indicated times (in minutes) samples were diluted and centrifuged through a sucrose cushion to separate cytosolic and nuclear fractions. A sample from the top of the cushion was taken for immunoblot analysis ("cytoplasm"). The cushion was aspirated, the pellet was washed and centrifuged, and the entire pellet ("nuclei") was loaded on SDS PAGE for subsequent immunoblotting with anti-XCdc14α antibodies. In vitro translated and 35S-labeled Xic1 was used as a positive control for nuclear formation and import (bottom panels). Xic1 was imported into the nucleus and destroyed by 55 minutes. Ubiquitinated forms of Xic1 can be seen in the nuclear fractions.