Activation of Notch-1 and Notch-2 by ligation with Jagged-1. HPCs were placed on 3T3-MSCV or 3T3-Jagged-1 fibroblasts for 5 hours; CD45⁺ cells were isolated and nuclear protein was extracted. CBF-1 binding to DNA was determined by EMSA. For blockade of CBF-1 binding 2 μg/lane nuclear protein was preincubated with specific antibodies against Notch-1 or Notch-1, for 30 minutes on ice prior to incubation with a α-³²P probe. Fifty-fold excess of unlabeled “cold” wild-type probe competitor in sample from cells incubated with Jagged-1 fibroblasts (lane 1); samples from cells cocultured with control (lane 2) and Jagged-1 fibroblasts (lane 3); blockade of CBF-1–binding activity with control antibody against mouse I-A^d, k (lane 4), antibody against Notch-1 (lane 5), antibody against Notch-2 (lane 6), and combination of antibodies against Notch-1 and Notch-2 (lane 7). Two experiments with the same results were performed.