Notch1 activation is incompatible with pDC development. (A) Serial dilutions of CD34⁺CD1a^-, CD34⁺CD1a⁺, and CD123^hiCD45RA⁺ cells were analyzed for expression of Notch1 mRNA by RT-PCR. Actin mRNA levels were measured as loading control (n = 3). (B) CD34⁺CD1a^- cells were cocultured for 3 hours with OP9-DL1, OP9-Jag1, or control cells. The presence of non-membrane-bound intracellular Notch1 (icNotch1) was analyzed by Western blot using an antibody recognizing the intracellular part of the Notch1 receptor.An antibody recognizing actin was used as loading control (n = 2). (C) CD34⁺CD1a^- cells were retrovirally transduced with icNotch1 or GFP control virus, cocultured on OP9 control cells, and analyzed for the presence of CD123^hiBDCA2⁺ pDCs after 1 week (n = 4), or (D) analyzed for the presence of CD4, CD8, and CD3 after 3 weeks (n = 2). Experiments representative of at least 2 experiments are shown.