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Fig. 1. The C-terminal Polo-box domain of Plk1 is responsible for its binding to endogenous CHO1/MKLP-1. (A) FLAG-tagged Plk1 constructs used in the transfection experiment. The hatched and filled bars indicate the positions of kinase domain (residues 53-305) and Polo box (residues 410-440), respectively. Stars indicate the positions of three point mutations within the polo box (W414F, V415A, L427A). (B,C) Cos-7 cells were transiently transfected with 10 µg of the indicated Plk1 constructs per 10-cm Petri dish. 30 hours after transfection, cells were treated with 100 ng ml–1 nocodazole for 16 hours and harvested. Cell lysates were subjected to anti-CHO1/MKLP-1 antibody immunoprecipitation, and anti-FLAG western blotting. As a control, the same quantities of cell lysates were incubated with Protein-A beads only. About 5% of the total cell lysates was used to assess expression. (D) Different amounts of Plk1 constructs (2.5 µg, 5 µg or 10 µg of DNA per 10-cm dish) were used for transfection as described above. Cell lysates were immunoprecipitated with anti-CHO1/MKLP-1 antibody, followed by anti-FLAG immunoblotting.
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