Truncated Notch1 transcripts and proteins in IN1C+ tumors. (A) Northern blot of total RNA (10 μg) from primary tumors and cell lines of the indicated genotypes using a probe for exon 34 (top). Methylene blue staining of 18S and 28S ribosomal RNA was used as a loading control (bottom). (B) RT-quantitative PCR of Notch1 transcripts, using primers to amplify exons 23-24 (encoding the extracellular EGF repeats 33/34) and exons 30-31 (encoding the first intracellular ankyrin repeat). Results were normalized to hypoxanthine-guanine phosphoribosyltransferase levels and to those of WT thymocytes, for which the exon 23-24 mRNA level was arbitrarily fixed at 1. Data represent the mean of 2 experiments. (C) Scheme summarizing the results of the 5′-RACE experiments performed on the T1 and T34 IN1C⁺ cell lines. The organization of the Notch1 transcript in the region of interest is shown, with the position of the S1, S2, and S3 cleavage sites, and the putative methionines that could be used for translation initiation. cDNAs identified by 5′-RACE are shown (see supplemental Figure 5 for sequence details). (D) Northern blot of Notch1 transcripts in poly(A)⁺ RNA (1.5 μg each) from the T1 cell line, hybridized with probes from the indicated exons/introns. E25(5′) corresponds to nt 4279-4741; E25(3′)/26 corresponds to nt 4758-5246; E30/31 corresponds to nt 5715-6179 (reference sequence NM_008714). E27b corresponds to a 628 nt region from intron 27 that includes the 113 nt sequence of exon 27b (see supplemental Figure 5). Autoradiograms for E25(3′)/26 and E30/31 were exposed for 18 hours; those for E25(5′) and E27b were exposed for 44 hours. Asterisks indicate transcripts initiating from 5′ promoters; black arrowheads indicate transcripts initiating from exon 25; white arrowheads indicate transcripts initiating downstream of exon 26 (likely in exon 27); white asterisks indicate transcripts containing exon 27b. (E) RT-PCR of exon 27b–containing transcripts in the indicated samples. cDNA was amplified using a forward primer located within exon 27b and a reverse primer from exon 31. The arrowhead indicates the correctly spliced transcripts; the asterisk indicates likely splicing intermediates that had not excised intron 27. (F) Western blot of Notch1 expression in total cell extracts from the T1 IN1C⁺ and T7 IN1C⁻ cell lines, cultured in the presence or absence of GSI for 3 days. The membrane was first analyzed with the Val1744 antibody, and then with the mN1A Ab. α-tubulin was used as a loading control. Long (10 minutes) and short (30 seconds) exposures are shown for the mN1A Ab. The lines between the top 2 panels indicate the positions of the molecular weight markers used to align the blots. Asterisks indicate the γ-secretase–cleaved proteins from the T7 cell line; arrowheads indicate the γ-secretase–cleaved proteins from the T1 cell line. Note that the ICN1 proteins in the T7 line do not completely disappear after GSI treatment, probably due to the increased stability of the truncated proteins in this cell line. All data are representative of > 2 independent experiments.