Fig. 2. Detection of XBub3 by immunoblotting and its co-immunoprecipitation with XBub1/XBubR1. (A) Immunoblot analysis of XBub3 in XTC cell lysates (XTC), interphase egg extracts (Int) or CSF egg extracts (M), using sheep and rabbit XBub3 peptide and full-length XBub3 antibodies. A doublet of bands in egg extracts and a single band in XTC cell lysates were specifically detected. (B) Immunoblot analysis of proteins immunoprecipitated from a denatured (TCA precipitated and re-natured) CSF egg extract using XBub3 antibody beads (XBub3 IP) or control beads without antibody. Both bands of the XBub3 were immunoprecipitated, although neither can be efficiently immunoprecipitated from a native extract (data not shown). (C) XBub1 and XBub3 co-immunoprecipitate. CSF egg extracts were incubated with beads alone or XBub1 beads and the immunoprecipitates were then treated with (+) or without (-) protein phosphatase ( pptase). Bound proteins were eluted in sample buffer, separated by SDS-PAGE and the Bub proteins were then detected by immunoblotting using rabbit anti-XBub1 antibody or sheep anti-XBub3 peptide antibody. (D) As in C, except that the XBub1 was further resolved by running a large 7.5% SDS polyacrylamide gel for 12 hours. (E) XBubR1 and XBub3 co-immunoprecipitate. CSF egg extracts (M) were immunoprecipitated with BUBR1 antibodies (BR1 IP) or beads alone. Samples of BubR1-depleted supernatant (BR1 sup) or beads alone supernatant (con sup) are shown. Bub proteins were detected by immunoblotting using rabbit anti-XBubR1 antibody or sheep anti-XBub3 peptide antibody.