Figure 6.

Cyclins accumulate and associate with Cdc2 in MAP kinase-activated extracts. Cycling extracts were supplemented with sperm chromatin, [³⁵S]methionine, and buffer (□) or Mos (▪). (A) Histone H1 kinase assays were performed on samples removed every 10 min. (B) Cyclin accumulation and Cdc2 tyrosine phosphorylation. Parallel samples were removed every 15 min and subjected to p13^suc1 agarose precipitation and SDS-polyacrylamide gel electrophoresis. Suc1-precipitated ³⁵S-labeled proteins were detected by autoradiography (upper). The cyclins exhibit an abrupt decrease in signal at mitosis (75 min) in the buffer-treated extracts while continuing to accumulate in the Mos-treated extracts. The lower half of the blot was probed with a phosphotyrosine antibody (middle), stripped, and reprobed with a Cdc2 antibody (lower). The level of phosphotyrosine increases over time in the Mos-treated extracts, and cycles in the buffer-treated extracts. Levels of cyclin, Cdc2, and Cdc2-PTyr were quantified by PhosphorImager scanning.