Immobilized Delta1^ext-myc specifically activated Notch signaling and effectively inhibited myeloid differentiation in the presence of fibronectin CH-296. (a) Culture plates were precoated either with anti-myc 9E10 antibody F(ab′)₂ to immobilize ligand (I-Delta) or with isotype-matched 31A antibody F(ab′)₂ (NI-Delta), with (+) or without (–) fibronectin fragment CH-296. CD34⁺CD38^– precursors (250 cells per well) were cultured for 2 weeks in serum-free medium containing SCF, FL, TPO, IL-6, and IL-3 (5GF) and either 1 μg/ml of Delta1^ext-myc or the same volume of control medium (Control), and analyzed by FACS for CD34 and CD14 expression. Data are representative of three independent experiments. Numbers in corners are the percentage of gated events within that quadrant. (b) CD34⁺ cells were cultured for 48 hours with 5GF, CH-296, and I-Delta1^ext-myc, NI-Delta1^ext-myc, or control medium, and expression of the Notch target gene HES-1 relative to that of GAPDH was assessed by quantitative RT-PCR. Expression of HES-1 mRNA was measured, and ratios were compared with those in control cells.