Retroviral constructs and transduction. (A) Structure of Notch1 and ICN and of the retroviral vector MSCV 2.2. (▪), hydrophobic leader; (▩), EGF-like repeats; (▨), LNR repeats; (‖▪‖), transmembrane domain; (▧), ankyrin repeats; Q, glutamine-rich region; P, PEST sequence. The MSCV LTR, IRES motif, and GFP gene of the MSCV 2.2 vector are indicated. (B) Flow cytometric analysis for GFP in HL-60–transduced cells. Contour plots represent fluorescence intensity for GFP on the x-axis and cell forward scatter on the y-axis. (C) Flow cytometric analysis for Notch1 on gated HL-60 and CD34⁺ GFP-positive cells. The polyclonal antibody directed against the intracytoplasmatic region of Notch1, T3, was used as described. Histograms are expressed as fluorescence intensity for Notch1 on the x-axis (log scale) and cell count on the y-axis. Superimposed are fluorograms with anti-GST control antibody on MSCV-GFP cells (left, filled curve), anti-T3 antibody on MSCV-GFP cells (middle curve, solid line), and anti-T3 antibody on MSCV-ICN/GFP cells (right curve, dotted line). Fluorograms with anti-T3 antibody on MSCV-GFP cells and uninfected cells were superimposable.