Fig. 2.
PAF15 is degraded in G1 by the anaphase-promoting complex. (A) PAF15 contains a KEN box that is highly conserved among its vertebrate homologs. (B) Wild-type and KEN mutant (K78A) PAF15 was expressed using the GPS vector in both 293T and HeLa cells. Cells were gaited on the basis of their cell cycle state after staining with Hoechst. Overlaid histograms of the EGFP/DsRed ratio for cells in G1, S, and G2/M phases are shown. (C) HeLa cells were arrested at the G2/M boundary using the CDK1 inhibitor RO3306. Following release, HA-tagged WT and KEN mutant PAF15 were followed by Western blot. Immunoblotting for cyclin B and vinculin provides a marker for cell cycle status and a loading control, respectively. (D) GPS-PAF15 was expressed in WT and Cdh1 null (−/−) MEFs. Cells were gaited on the basis of cell cycle state after staining with Hoechst. Overlaid histograms of the EGFP/DsRed ratio for cells in G1, S, and G2/M phases are shown. (E) HeLa cells were treated with siRNA targeting firefly luciferase (FF) as a negative control or Cdh1. Seventy-two hours after transfection cells were collected for Western blot and analyzed with antibodies to PAF15, geminin, Cdh1, and vinculin (loading control).