Figure 2.

Active mBub1 kinase associates with mBub3. (A) Recombinant mBub1 and mBub3 coimmunoprecipitate. Lysates from insect cells coexpressing HIS₁₀HA-Bub1 and HIS₁₀HA-Bub3 or expressing HIS₁₀HA-Bub1 alone were analyzed by using antipeptide antibodies against either mBub1 (lanes 2, 3, and 6) or mBub3 (lanes 4 and 5). A 20-fold molar excess of nonspecific (lanes 2 and 4) or specific peptide (lanes 3 and 5) were added as specificity controls. (B) Protein kinase activity of mBub1. Immune complexes containing HIS₁₀HA-Bub1 and HIS₁₀HA-Bub3 (lanes 7–10) or HIS₁₀HA-Bub1 alone (lane 11) were prepared as in A and incubated with ³²P-γ-ATP at room temperature for 30 min in kinase buffer before analysis by autoradiography. (C) hBub3 is present at similar levels in interphase and mitotic HeLa cells. Fifty micrograms of extract from interphase (lanes 12 and 14) or nocodazole-treated (lanes 13 and 15) cells were analyzed by Western blotting using anti-Bub3 or anti-cyclin B antibodies.