JME mutants do not affect the stability of the LNR-HD complex. (A) Schematic representation of the soluble LNR-HD complex encoded by the NOTCH1 minigene. In addition to the LNR repeats (green), the N-terminal (HD-N) and C-terminal (HD-C) subunits of the HD domain (blue), and the extracellular juxtamembrane region (JM), this construct contains an N-terminal FLAG tag and a C-terminal HA tag. (B) Immunoprecipitation immunoblot analysis of NOTCH1 minigene protein products recovered from conditioned media of 293T cells transfected with empty plasmid (control), a NOTCH1 wild-type minigene (WT), a NOTCH1 minigene containing the Jurkat JME 17 amino acid insertion (Jurkat JME 17), and a NOTCH1 minigene containing the artificial VSV-G-P JME 14 amino acid insertion. (C) Immunoprecipitation immunoblot analysis of NOTCH1 minigene protein products from a NOTCH1 wild-type minigene (WT) and NOTCH1 minigene containing the Jurkat JME 17 amino acid insertion (Jurkat JME 17) incubated with increased concentrations of urea to test the stability of the LNR-HD domain complex.