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Fig. 2. The stalk domain of CHO1/MKLP-1 is responsible for its binding to endogenous Plk1. (A) HA-tagged CHO1/MKLP-1 constructs used in the transfection experiment. (B) Cos-7 cells were transiently transfected with CHO1/MKLP-1 constructs as indicated. 30 hours after transfection, cells were treated with 100 ng ml–1 nocodazole for 16 hours and harvested. About 5% of total soluble cell lysates (leftmost six lanes) or immunoprecipitates with anti-Plk1 rabbit polyclonal antibody from the transfected cells (rightmost six lanes) were subjected to western blotting with anti-HA antibody. The positions of molecular size markers are indicated at the left. Stars indicate non-specific proteins that cross-react with the anti-HA antibody. (C) Colocalization of the stalk domain of CHO1/MKLP-1 with Plk1 at the mid-body. GFP-fused CHO1/MKLP-1 domains were transfected into HeLa cells. 30 hours after transfection, cells were treated with 50 ng ml–1 nocodazole for 8 hours. Mitotic cells were mechanically shaken off and collected. After nocodazole was washed away over a 1 hour period with ice-cold DMEM, the cells were mixed with prewarmed medium and replated onto polylysine-coated coverslips for 75 minutes. Plk1 was stained with anti-Plk1 mouse monoclonal antibody, followed by Cy3-conjugated secondary antibody. DNA was stained with DAPI. Scale bar, 10 µm.
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