FIG. 2.

The Tsg24 protein binds to murine centromeres in a cell cycle-independent manner. Mitotic or interphase murine Swiss-3T3 cells were fixed with methanol-acetone (50:50 [vol/vol]), triple stained with different combinations of antibodies, and analyzed by indirect immunofluorescence microscopy. Rows 1 and 2 show mitotic cells labelled with the anti-Tsg24 antibody (1:20), a CREST antiserum (1:500), and Hoechst 33258. Rows 3 to 5 show interphase cells and late-S-phase cells triple stained with the anti-Tsg24 antibody (1:20) or a preimmune serum, a monoclonal anti-PCNA antibody (1:500), and Hoechst 33258. The anti-PCNA antibody labels early- (e) and late-S-phase (l) cells. Late-S-phase cells were also analyzed at a higher magnification (rows 4 and 5). The secondary antibodies used were the same as in Fig. 1, except that a rhodamine-conjugated goat anti-mouse IgG was used to label the anti-PCNA antibody. Fixation of cells with paraformaldehyde gave identical results but weaker signals. Staining of murine L cells gave identical results.