Effect of Notch ligands on downstream targets of Notch signaling. (A) Effect of ICN1 and ICN2 on differentiation of DCs. HPCs were transduced with either ICN1 or ICN2 using retroviruses as described in “Materials and methods” and differentiated to DCs with GM-CSF for 7 days. After that time, cells were collected and the phenotype of GFP-positive cells was evaluated by flow cytometry. The results of 4 experiments are shown. (B) CBF-1 binding activity. HPCs were cultured on a monolayer of different 3T3 fibroblast cell lines for 8 hours. Nuclear protein was extracted and used for evaluation of CBF-1 binding to ³²P-labeled specific DNA probe in EMSA. (C) CBF-1 reporter luciferase activity induced by Notch ligands. HPCs from Balb/c mice were infected with control KA9 virus or CBF-1 reporter virus twice on day 0 and day 1 with a 16-hour interval. After second infection, cells were cultured for 48 hours. To activate Notch signaling, infected HPCs were cultured for 8 hours on 3T3 fibroblasts expressing different ligands. Luciferase activity was normalized on protein concentration. Mean ± SE of 3 performed experiments is shown. (D) HPCs were electroporated with pGL3 or NF-κB reporter plasmids and 40 hours later were placed on top of a monolayer of different fibroblast cell lines. M indicates 3T3-MSCV; J, 3T3-Jagged-1; E, 3T3-EF; and D, 3T3-Delta-1. After overnight incubation, cells were collected, CD45⁺ cells were isolated using magnetic beads, whole-cell lysates were prepared, and luciferase activity was measured as described in “Materials and methods.” The results were normalized for protein concentrations. (E) HPCs were cultured for 72 hours on different cell lines, whole-cell lysates were performed, and protein level was evaluated by Western blotting using indicated specific antibodies. Data represent the average ± SE of 3 experiments.