Fig. 4.

Influence of depletion of Plk1 on levels of Emi1 in mitotically arrested HeLa cells. (A) Effects of transfection with siRNA duplex directed against Plk1 on levels of Plk1, cyclin B, and Emi1 proteins. HeLa cells were either mock-treated or transfected with siRNA directed against human Plk1, as described in Methods. Where indicated, nocodazole (Noc, 0.5 μg/ml) was added 16 h before collection of cells. Cells were collected 24 h after transfection, and samples were immunoblotted with antibodies directed against Plk1 (Top), cyclin B (Middle), and C-terminal peptide of Emi1 (Bottom). (B) Specificity of anti-Emi1 antibody. Extracts from asynchronous (As) or nocodazole-arrested cells and 0.1 ng of recombinant Emi1 (Rec) were separated on 10% SDS/polyacrylamide gels, transferred to nitrocellulose, and blotted with an antibody directed against a C-terminal peptide of Emi1 (1 μg/ml) in the absence (lanes 1–3) or presence (lanes 4–6) of C-terminal peptide (0.1 μg/ml). Asterisks denote crossreacting proteins; the number on the right indicates the position of molecular mass marker protein (kDa).