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Fig. 2. Cdh1 is required for the destruction of Aur-A in HeLa cells. (A) HeLa cells were infected with retrovirus encoding His₆-tagged N-terminal dominant negative fragment of Cdh1 [Cdh1(1-125)] or an empty retrovirus control. 24 hours after infection, cells were lysed and processed for western blotting with antibodies against His₆ or Aur-A, as indicated, or for propidium iodide staining and flow cytometry. (B) HeLa cells were transfected with or without pcDNA3-FLAG-CUL1(1-452). 48 hours after transfection, cells were lysed and processed for western blotting with antibodies against FLAG, p27 or Aur-A, as indicated. In both A and B, the Aur-A blot was stripped and reprobed with anti-Cdc2 antibody as a loading control. (C) IVT Myc tag, Myc-Cdh1 or Myc-Cdc20 were incubated with anti-Myc beads and washed extensively. Beads were then incubated with [³⁵S]-labeled IVT GFP or GFP/Aur-A variants, as indicated, followed by washes and elution into SDS sample buffer. Samples were analysed by PAGE and autoradiography alongside 10% of the input IVT product, and bands quantified by phosphorimager analysis.

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