Notch1 mutations and Notch-dependent proliferation of C3G-F⁺ SPA-1^−/− T-ALL cell lines. (A) T-ALL cell lines (Wo1, Wo2, and Wo3) established from the thymi of C3G-F/SPA-1^−/− HPC recipients as well as normal thymocytes were lysed and immunoblotted with indicated antibodies. Rap1GTP and Ras GTP were detected by a pull-down assay. F indicates full-length Notch; TM; transmembrane form; and NICD, Notch intracellular domain. Note smaller NICD1 size in Wo1 line (solid arrowhead) than in normal thymocytes (open arrowhead). (B) T-ALL cell lines were cultured in the presence of various concentrations of DAPT for 48 hours, and the expression of indicated proteins was detected by immunoblotting (left upper panel). They were also cultured in the absence (circles) or presence of 0.3 μM (triangles) or 1 μM (rectangles) DAPT, and the viable cell numbers were determined by MTT assay at days 2 and 3 (left lower panel). Means and SE of triplicate cultures are indicated. T-ALL cells were cultured in the presence or absence of 1 μM DAPT for 2 days and analyzed for DNA contents (right panels). (C) Notch1 cDNAs of approximately 1.7-kb upstream of PEST region (from nt 6212 to nt 7973) were cloned from 3 independent T-ALL lines, and more than 5 subclones of each were sequenced. Insertions detected in more than 3 subclones were considered to represent genuine mutations and are indicated (top panel). Deduced amino acid sequences are also indicated (bottom panel). An underline indicates the PEST domain, and an asterisk marks the stop codon. T2512 required for the binding of FBW7 ubiquitin ligase is indicated by a dot.