FIG. 2.

Mitotic degradation of hTK1 requires functional Cdh1. (A) HeLa cells were transfected with pFLAG-Cdc20(1-120), pFLAG-Cdh1(1-125) or empty vector (Mock) together with pEGFP as an internal control. Following transfection for 24 h, cells were synchronized as described in the legend to Fig. 1. One set of cells during release from the mitotic arrest was treated with LLnL (100 μM) for 6 h. Expression of hTK1, Cdc20(1-120), Cdh1(1-125), and GFP was analyzed by Western blotting with specific antibodies (Ab). (B) LM TK⁻ cells were transfected with 1 μg of pCDNA3.1-hTK1 and different amounts of pHA-Cdh1 as indicated. Empty vector was added to have a final 3 μg of total DNA for transfection. Cells were treated without (−) or with (+) LLnL (100 μM) for 6 h before harvesting. HA-Cdh1, hTK1, and β-tubulin were detected by Western blot analysis. (C) LM-TK− cells transfected with pCDNA3.1-hTK1 and different amounts of pHA-Cdc20 as indicated were analyzed as described above. (D) HeLa cells were transfected without (−) or with (+) 0.8 μg of duplex siRNA against Cdh1 as described in Materials and Methods. After transfection at different time points, cells were extracted for Western blot analysis. (E) Scheme of the synchronization procedures for Cdh1 siRNA transfection experiment in HeLa cells (upper panel). Cell extracts were prepared after transfection at different phases as indicated and analyzed as described above.