Figure 6. Prc1 is required for Plk1-mediated stimulation of the HsCyk-4-Ect2 complex.

(A) Control HeLa cells, or those transfected with siRNA to deplete endogenous Ect2 and Prc1, were synchronized in S-phase with thymidine or in prometaphase with nocodazole. Cells were treated with DMSO or 22.5 µM purvalanol A. Lysates and Ect2-BRCT–bound fractions were separated on SDS-PAGE and probed with antibodies to HsCyk-4, α-tubulin, Ect2, and Prc1. Lysate represents approximately 5% of input. (B) HeLa cells were either mock transfected or transfected with siRNA to deplete Prc1, and synchronized in prometaphase with nocodazole. Cells were released from the prometaphase block by either addition of 22.5 µM purvalanol A or replacement with fresh medium and harvested 60 min post-release. Lysates and HsCyk-4 immunoprecipitates were separated on SDS-PAGE. Blots were probed with antibodies to HsCyk-4 and phospho-Ser170. (C) The indicated stable cell lines transfected with siRNA to deplete endogenous HsCyk-4 and Prc1, as indicated, were synchronized in prometaphase with nocodazole, at which point cells were treated with 22.5 µM purvalanol A. Lysates and Ect2-BRCT–bound fractions were separated on SDS-PAGE and probed with antibodies to HsCyk-4, Prc1, and α-tubulin. Lysate represents approximately 5% of input. Asterisks indicate the position of cross-reacting species in recombinant CBD-Ect2-BRCT recognized by Prc1 antibodies. (D) HeLa cells were synchronized with 0.04 µg/ml nocodazole, at which point either DMSO or nocodazole was added to a final concentration of 5 µg/ml in the presence of 22.5 µM purvalanol A. Lysates and Ect2-BRCT–bound fractions were separated on SDS-PAGE and probed with antibodies to HsCyk-4, cyclin B1, and α-tubulin (upper gels). Lysate represents approximately 5% of input. HeLa cells, synchronized and treated as above, were fixed and stained with antibodies to HsCyk-4 and α-tubulin, and DNA was stained with DAPI (lower images).