Validation of a NICD2-specific antibody. (A) Schematic showing a “headless” form of NOTCH2 (ΔE-N2) retaining the leader peptide (L) and lacking the extracellular NRR that is sensitive to successive cleavages by ADAM10 (ADAM) and γ-secretase (GS), releasing NICD2 from the transmembrane domain (TMD) and generating an N-terminal neoepitope. (B) Western blot showing specificity of NICD2 antibody. Whole-cell extracts were prepared from (i) 293T cells transfected with empty vector or ΔE-N2 complementary DNA (cDNA) and treated with dimethyl sulfoxide (DMSO; vehicle control) or γ-secretase inhibitor (1 μM compound E; γsecretase inhibitor [GSI]) posttransfection, and (ii) B-cell lymphoma cell lines with (Ri-1) or without (SUDHL4, WSU-DLCL2, OCILy3, and OCILy10) gain-of-function mutations in NOTCH2. Note NICD2 staining is suppressed by GSI treatment in transfected 293T cells and is also observed in NOTCH2-mutated Ri-1 cells. (C-G) Representative images of NICD2 immunostaining in sections prepared from FFPE tonsil (C), lymph node (D), normal spleen (E), spleen with reactive marginal zone expansion (F), and spleen involved by NOTCH2-mutated SMZL (G). The sections in panels C-G were counterstained with hematoxylin (blue) after immunostaining using a method that produces a brown color. Tonsil and lymph node sections show only weak nuclear staining in subsets of lymphocytes in the perifollicular regions. Spleen sections show moderate nuclear staining of a subset of lymphocytes within marginal zones. Arrows in C-F highlight the position of cells that are shown at high power in the insets. The NOTCH2-mutated SMZL shows diffuse, strong nuclear staining in neoplastic cells. Original magnification, ×200; inset, ×400. ANK, ankyrin repeat domain; PEST, PEST degron domain; RAM, RBP-Jκ/CBF1-associated module.