Cul4A promotes the proteasome-dependent degradation of p27. (A) Proliferating G1E-ER4 cells were treated with proteasome inhibitor (5 or 10 μM MG132) for 4 hours. Cells were harvested, protein lysates prepared, and 10 μg total protein for each sample was analyzed by immunoblot probed with antibodies that recognize p27 or actin. Actin was used as a loading control. Representative results of 2 independent experiments are shown. (B) Cul4A-HA and empty vector control cells were induced to differentiate for 24 hours, 3 × 106 cells/100-mm dish were treated with 10 μg/mL cycloheximide for the indicated times, cells were harvested, lysates were prepared, and 10 to 30 μg total lysate for each sample was analyzed by immunoblot and probed with anti-p27 or anti–β-actin antibodies. Actin was used as a loading control. For Cul4A-HA (▴) and empty vector control cells (▪), the amount of p27 at each time point was normalized to the amount of p27 at 0 hours (100%). Because significantly less p27 is present in Cul4A-HA cells compared to the control, 2 different exposures of the same anti-p27 immunoblot are shown. Representative results from 2 independent experiments are shown. (C) For lanes 1-4, Cul4A-HA cells were induced to differentiate for 24 hours, cells were harvested, protein lysate was prepared, and for each sample, 1 mg total protein lysate was precleared with protein A and protein G Sepharose beads, followed by immunoprecipitation with anti-p27, anti-Cul4A, or anti-HA antisera (all from rabbit) and incubation with protein A and protein G Sepharose beads. Immunoprecipitated proteins were eluted with sample buffer and analyzed by immunoblot probed with antibodies that recognize HA, Cul4A, or p27. Immunoprecipitation with protein A and protein G Sepharose beads alone was used as a negative control. For lanes 5-9, G1E-ER4 cells were induced to differentiate for 24 hours, protein lysate was prepared, immunoprecipitations with anti-HA, anti-p27, or anti-Cul4A antisera were performed as described for lanes 1-4. Immunoprecipitated proteins were eluted with sample buffer and analyzed by immunoblot probed with antibodies that recognize Cul4A or p27. Immunoprecipitations with either normal rabbit IgG, protein A and protein G Sepharose beads alone, or rabbit anti-HA antiserum (antiserum against an unrelated protein in these cells) were used as negative controls, all of which failed to immunoprecipitate either p27 or Cul4A, indicating that the anti-p27 and anti-Cul4A antibodies interact specifically with their respective antigens. Representative results from 3 independent experiments are shown.
