NOTCH1 protein expression in T-cell acute lymphocytic leukemia (T-ALL) cell lines with t(9;14). (a) Western analysis of T-ALL cell lines: NOTCH1-ANK domain antibody (Ab) to mN1A (top), bTAN20 Ab to transcriptional activation domains (TADs) (middle) and Ab8925 (activated NOTCH1) to TM/RAM (regulator of amino acid metabolism) (bottom). Ab8925 epitopes include a non-specific band (star) and require prior γ-secretase exposure (manufacturer's data). Note the weak TM/RAM epitopes in SUP-T1 NTM, which are attributable to impaired γ-secretase cleavage. (b) Western analysis of full-length NOTCH1 at longer exposure: note the faint (HD-MAR/HT-1) or absent (SUP-T1) full-length NOTCH1 bands, implying translocation-driven expression. Western blots were performed after lysis with 50 μl of RIPA buffer (1 μl aprotinin (1 mg/ml), 5 μl PMSF (20 ng/ml) and 50 μl 2 × SDS). Antibodies used: NOTCH1 (mN1A; BD Biosciences, San Diego, CA, USA, bTAN-20; Hybridoma Bank, Iowa City, IA, USA and Ab8925; Abcam, Cambridge, UK). Loading was checked using Ponceau dye and anti-human-MSH6 Ab (Santa Cruz Biotechnology, Heidelberg, Germany).