(A) Expression of E2F-4 in human primary CD34⁺ hematopoietic cells. Purified CD34⁺ cells were induced to proliferate by the addition of cytokines as previously described in Materials and Methods. Samples were taken at the times shown and western blotting was performed using a specific anti-E2F–4 antibody. At 0 hours, the 68-kD form of E2F-4 predominates, but as the cells proliferate, the lower forms appear with the complete disappearance of the 68-kD form by 72 hours. A longer exposure of the same blot shows that there is little or no 68-kD form present in the 72- and 96-hour samples. (B) Dephosphorylation of E2F-4 in CD34⁺ cells by lambda phosphatase. A total of 4 μg of CD34⁺ whole cell lysate made 1 day after addition of cytokines was incubated for 1 hour with 500 U of lambda phosphatase. This was then added to sample buffer and separated by SDS-PAGE and probed with an anti-E2F–4 antibody. Dephosphorylation of the 68-kD form can be seen by the appearance of a lower, faster running band. Day 0 (quiescent) and day 4 (proliferating) samples are shown for comparison.