JME mutants are sensitive to inhibition of the γ-secretase complex and require S2 cleavage for activation. (A) Western blot analysis of activated NOTCH1 (ICN1) levels in Jurkat cells treated with CompE (100 nM) for 24 hours shows inhibition of NOTCH1 processing and clearance of activated intracellular NOTCH1. (B) Quantitative RT-PCR analysis of HES1 expression in Jurkat cells treated with CompE (100 nM) or vehicle only (DMSO). (C,D) CSL luciferase reporter assay (C) and Western blot analysis (D) of ICN1 activity and expression in cells expressing NOTCH1 JME mutants treated with vehicle (DMSO) or a γ-secretase inhibitor (CompE, 100 nM). (E,F) Luciferase reporter assay with a CSL reporter construct shows effective inhibition of NOTCH1 signaling from the NOTCH1 JURKAT JME 17 allele (E) and the NOTCH1 VSV-G JME 14 allele (F) upon mutation of the S2 cleavage site. Error bars represent SD.