Cell cycle–dependent regulation of DDA3 by ASB7. (A) Stabilization of DDA3 in S phase by knockdown of ASB7. Two independent siRNAs (#3 and #5) targeting ASB7 were transfected into HeLa cells and synchronized in S phase. The cells were released and harvested at the indicated times. The lysates were subjected to Western blotting with antibodies against DDA3, ASB7, Aurora A, cyclin E, or Hsp90. (B) The intensities of DDA3 bands in A were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. (C) Knockdown of ASB7 had no effect on the stability of DDA3 in M phase. Two independent siRNAs (#3 and #5) targeting ASB7 were transfected into HeLa cells and synchronized in M phase. The cells were released and harvested at the indicated times. The lysates were subjected to Western blotting with antibodies against DDA3, ASB7, Aurora A, p27, or Hsp90. (D) ASB7-dependent destabilization of DDA3 in S phase. Two independent siRNAs (#3 and #5) targeting ASB7 were transfected into HeLa cells and synchronized in the G1, S, or M phase. Nocodazole or taxol was used to synchronize cells in M phase. The cells were then exposed to 50 µg/ml CHX for 1 or 2 h. The lysates were subjected to Western blotting with antibodies against DDA3 or Hsp90. (A, C, and D) Hsp90 is shown as a loading control. (E) The intensities of DDA3 bands in D were normalized to those of the corresponding Hsp90 bands and plotted as a ratio of the normalized value of control cells at 0 h. For all graphs, *, P < 0.05; **, P < 0.01. Data represent the mean ± SD of three independent experiments.