M1727 serves as the translational start site in aberrant type 1 and type 2 Notch1 transcripts. (A) Schematic showing the positions of codons M1616 (M1), M1659 (M2), and M1727 (M3) within type 1 and type 2 Notch1 transcripts relative to the full-length Notch1 polypeptide. (B) Western blot analysis of 293 cells transiently transfected with empty pcDNA3, type 1 transcript cDNA (corresponding to nucleotides 4438-7665 of murine Notch1), type 1 transcript cDNAs bearing M to L point mutations in codons 1616, 1659, or 1727, type 2 transcript cDNA from SCID.adh cells, or type 2 transcript cDNA with an M to L point mutation in codon 1727. Blots were stained with antibodies specific for activated Notch1 (V1744 antibody, Cell Signaling Technology) or β-actin. (C) Notch reporter gene studies conducted in U2OS cells transiently transfected in triplicate with equivalent amounts of a CSLx4 firefly luciferase reporter gene, an internal Renilla luciferase control gene, and empty pcDNA3 vector or pcDNA3 vectors containing the indicated cDNAs. The left-hand panel compares the activities of a murine type 1 Notch1 transcript; a murine type 1 Notch1 transcript containing a M1727L mutation; a murine type 1 Notch1 transcript containing a stop mutation introduced at codon 2415 (ΔP) that deletes the C-terminal 116 amino acids of murine Notch1; a full-length human NOTCH1 cDNA containing a weakly activating NRR mutation, L1601P, with and without a ΔP frameshift mutation at codon 2473 that deletes the C-terminal 83 amino acids of human Notch1; and a full-length human NOTCH1 cDNA containing a strongly activating in-frame insertion in the NRR designated P12, with and without a ΔP frameshift mutation at codon 2473. The right-hand panel compares the activities of murine type 1 and type 2 transcripts with a human NOTCH1 cDNA bearing a L1601P deletion. Each cDNA was tested in the presence and absence vehicle (dimethyl sulfoxide [DMSO]) or vehicle plus the GSI DAPT (10μM). After normalization to an internal Renilla luciferase control, firefly luciferase activities were expressed relative to the empty vector control, which was arbitrarily set to 1. Error bars represent SD. The results shown are representative of 2 independent experiments.