NOTCH1 JME mutants show high levels of NOTCH1 signaling. (A) Western blot analysis of 293T cells expressing FLAG-tagged forms of wild-type (WT) and NOTCH1 mutant alleles. NOTCH1 HD L1600P is class 1 HD mutant; ΔPEST encodes a C-terminal truncation of NOTCH1 lacking a PEST domain; L1600P⁺ΔPEST is a double-mutant allele of NOTCH1 harboring both an HD mutation and a PEST domain truncation; and Jurkat JME 17 is the NOTCH1 exon 28 JME mutant allele identified in Jurkat cells. Total NOTCH1 levels were detected with anti-FLAG antibodies (top panel). ICN1 protein was detected with NOTCH1 Val1744 antibody, which specifically recognizes the γ-secretase cleaved activated form of NOTCH1. Luciferase is shown as transfection/loading control. (B) Transactivation activity of HD, ΔPEST, HD-ΔPEST, and Jurkat JME 17 NOTCH1 alleles in a CSL luciferase reporter assay. Reporter activity is shown as fold change compared with wild-type NOTCH1 (WT). (C) Transactivation activity of artificially generated NOTCH1 JME alleles. NOTCH1 constructs expressing juxtamembrane insertions of 5 to 14 amino acids corresponding to the VSV-G epitope as well as the Jurkat JME 17 allele were tested in a CSL luciferase reporter assay. Reporter activity is shown as fold change compared with wild-type NOTCH1 (WT). Error bars represent SD.