The NOTCH1 del-N mutant shows high levels of NOTCH1 signaling and is sensitive to inhibition of the γ-secretase complex. (A) CSL luciferase reporter assay of ICN1 activity in HeLa cells transfected with empty vector (control) or expressing wild-type NOTCH1 (NOTCH1), NOTCH1 del-N deletion mutant (NOTCH1 del-N), NOTCH1 HD ΔPEST (HDΔPEST), NOTCH1 Jurkat JME17 (JME17), or NOTCH1 P12 (P12) mutant alleles. Reporter activity is shown as the fold change compared with wild-type NOTCH1. Error bars represent SD. P values were derived from Student t test. (B) Western blot analysis of 293T cells expressing empty vector (Control) or FLAG-tagged forms of wild-type and del-N mutant NOTCH1 alleles. ICN1 protein was detected with the NOTCH1 Val1744 Ab, which specifically recognizes the γ-secretase–cleaved activated form of NOTCH1. Total NOTCH1 levels were detected with an anti-FLAG Ab. Actin is shown as a loading control. (C) Transactivation activity of NOTCH1 del-N, HDΔPEST, Jurkat JME17, and P12 NOTCH1 alleles in a CSL luciferase reporter assay in HeLa cells treated with vehicle (DMSO) or a γ-secretase inhibitor (compound E, 250 nM). Reporter activity is shown as the fold reduction compared with vehicle-treated cells for each allele. Error bars represent SD. (D) Western blot analysis of activated NOTCH1 (ICN1) levels in 293T cells treated with compound E (250nM) for 48 hours shows inhibition of NOTCH1 processing and clearance of activated intracellular NOTCH1. ICN1 protein was detected with NOTCH1 Val1744 Ab, total NOTCH1 levels were detected with anti-FLAG Abs, and actin is shown as a loading control.