Identification of internal tandem duplications in the extracellular juxtamembrane domain of NOTCH1 in T-ALL. (A) Western blot analysis of T-ALL cell lines lacking HD and PEST domain mutations in NOTCH1 demonstrating high levels of activated NOTCH1 in the Jurkat cell line. ICN1 levels were detected with the NOTCH1 Val1744 antibody. Tubulin is shown as loading control. (B) Sequence analysis of the NOTCH1 transcripts showing the mutation identified in the Jurkat cell line. The wild-type NOTCH1 transcript sequence is shown at the bottom and inserted sequences on top. S2 and S3 indicate the sites for ADAM metalloprotease and γ-secretase cleavage, respectively. Duplicated nucleotides are shown in italics and underlined in the wild-type sequence. The common 4 amino acids (QLHF) present in all JME mutants identified in this series are highlighted in yellow. (C) PCR amplification of exon 28 of NOTCH1 and adjacent intronic sequences. Three samples corresponding to Jurkat cells and 2 independent primary T-ALL cases show bands of increased size compared with the control genomic DNA indicating the presence of insertions in the extracellular juxtamembrane domain of NOTCH1.