Nuclear NICD wt is stabilized in HG3 (NICDwt/152) cells and samples from patients with CLL with heterozygote 152 mutations.NOTCH1 152 A>G mutation in HG3 11 (A) and HG3 149 (B) was introduced by CRISPR/Cas9 (see supplemental Figure 9). After nuclear extraction, significantly higher intensities of endogenous nuclear NICD wt were detected by WB and the following quantification compared with HG3 wt. − indicates the absence of corresponding chemicals, and + denotes the presence of respective chemicals. Quantifications were performed by ImageJ. Mean ± SD; n = 4 in panel A and n = 7 in panel B; 2-tailed unpaired t test. Blots are representative of at least 3 blots. (C) Analyzing NICD signals from lysates of NOTCH1 wt (lanes 1-5), coding NOTCH1 ΔCT mutated (lanes 6-10) and NOTCH1 152 mutated (lanes 11-15) patients with CLL. Stabilization of truncated NICD (ΔCT patients) and even more evident NICD wt from NOTCH1 152 mutated patients can clearly be detected. Lysates from transfected NICD proteins, missing the amino-terminal valine 1754 residue and expressed in NOTCH1 KO HEK 29324 cells, served as a negative control (lanes 16-22). Lysates from DeltaMax stimulated HG3 cells29 (lane 23) and MO1043 cells expressing a truncated NICD served as positive controls for the antibody. A longer exposure blot is shown (middle). The α-NOTCH1∗ antibody (bottom) was used as a general NOTCH1 expression control.
