Figure 7.

Checkpoint complexes in nocodazole-arrested HeLa cells. (A) Mitotic checkpoint was activated in HeLa cells by a thymidine-nocodazole arrest. Cell lysates were fractionated through a Resource Q column and fractionation profiles of BubR1, Cdc20, APC2, and Mad2 were determined by immunoblotting. (B) Fractions from peak A were pooled, concentrated, and further fractionated by a Superose 6 column. Fractionation profiles of BubR1, Bub3, Cdc20, Mad2, and Mad1 were determined by immunoblotting. Arrowhead, a partial degradation product of Mad1. (C–E) Fractions 25, 29, and 37 from the Superose 6 column were immunoprecipitated with anti-Mad2 (C), anti-Cdc20 (D), and anti-BubR1 (E) antibodies, followed by Western blot analyses with antibodies against various checkpoint proteins.