NICD 152 expression dysregulates the regular NICD wt degradation process. (A) Co-IP performed in HEK 293 wt and NOTCH1 KO HEK 293 cells²⁴ to exclude an interference with endogenous NOTCH1. Ubiquitination of NICD wt was decreased after gradual increase of spliced NICD 152, which was verified by quantification of NICD wt ubiquitination signals with ImageJ, illustrated in the lower panel. Mean ± SD; n = 6; Kruskal-Wallis test followed by Dunn multiple comparison test for comparisons in HEK 293 wt; ordinary 1-way ANOVA test followed by Šídák multiple comparison test for NOTCH1 KO HEK 293. Active endogenous nuclear NICD wt is stabilized in HEK 293 wt (B) and HeLa wt (C) by overexpression of NICD 152. After nuclear extraction, in the presence of overexpressed NICD 152, signals of endogenous active nuclear NICD wt were stronger than in the group transfected with empty vector (pcDNA3). Quantification of WBs (right) reveals that intensities of endogenous active nuclear NICD wt in HEK 293 wt and HeLa wt after NICD 152 overexpression were significantly higher than in empty vector control group. Quantification of WBs was performed by ImageJ. Mean ± SD; n = 18 in panel B and n = 13 in panel C; 2-tailed unpaired t test. − indicates the absence of corresponding proteins or constructs, + denotes the presence of respective proteins or constructs, and ∗ represents the heavy chains of Flag antibodies. Each blot is a representative example of at least 3 blots.