Characterization of a specific antibody able to detect spliced NICD 152/143. (A) Carboxy-terminal protein sequences of NICD wt and spliced NICD variants (UniProt: P46531). The purple area indicates wt C-terminal amino acids, and the dark blue areas indicate aberrant C-terminal amino acids from spliced variants. Immunogen sequences used to produce a NICD 152/143 specific antibody are denoted in pink type. (B) Specificity test of the generated α-NICD 152 antibody. The α-NICD 152 from an immunized rabbit only detects NICD 152/143 proteins overexpressed in NOTCH1 KO HEK 293 cells.24 Spliced NICD 152/143 from NICD+3′UTR constructs were also detected by the α-NICD 152; however, splicing efficiency of NICD+3′UTR 143 is dramatically lower than NICD+3′UTR 152. The α-NICD 152 did not show any affinity to endogenous proteins in different cell lines; minus signs represent untransfected samples. (C) The α-NICD 152 antibody was able to detect NOTCH1 152 protein in patients with CLL harboring the corresponding mutation after IP but not in NOTCH1 wt and NOTCH1 ΔCT patients. A NOTCH1∗ (see supplemental Figure 4) antibody was used as a control. Asterisks represent the heavy chains of antibodies. Samples after precipitation were loaded for WB, and α-tubulin was used for protein abundance control of patient samples. IP, immunoprecipitation.
