Cul4A overexpression promotes proliferation in proerythroblasts. (A) Protein lysates were prepared from empty vector control or Cul4A-HA cells, and 10 μg of each was analyzed by immunoblot probed with anti-HA monoclonal antibody (top), anti-Cul4A antiserum (middle), or antiactin monoclonal antibody (bottom). The various forms of Cul4A are indicated with arrows on the right. An immunoblot representative of 3 independent experiments is shown. (B) Cul4A-HA and empty vector control cells were plated in triplicate (2.5 × 10⁴ cells/well) and cultured in 96-well plates in the presence of 2 U/mL Epo and 50 ng/mL SCF for 48 hours, incubated with tritiated thymidine for 8 hours, and incorporated tritiated thymidine was measured. The mean incorporated tritiated thymidine of triplicate samples was determined for each experiment, and the means (cpm ± SEM) of these values from 3 independent experiments are graphed. *P = .04. (C) Cul4A-HA and empty vector control cells were plated at 2.5 × 10⁵ cells/mL, grown for 24 hours, and the proportion of cells in each phase of the cell cycle was determined by propidium iodide staining and flow cytometric analysis. The means (± SEM) of 3 independent experiments are graphed. For cells in G₁ and S phase, *P < .05.