FIGURE 1.

Notch target genes are constitutively expressed in Ikaros-deficient leukemia cell lines that express ICN. A, Western blot analysis was used to assay for the presence of ICN in whole cell lysates prepared from Ikaros deficient T leukemia cell lines. Blots were probed with Abs directed against ICN that has been properly cleaved at val1744 (expected mobility: 110kD). Blots were also probed with anti-actin Abs as a loading control. B, Summary of sequence analysis of Notch1 using cDNA (PEST) or DNA (HD) from Ikaros deficient leukemia cell lines. HD = heterodimerization domain (exon 26 and exon 27). PEST = Proline, Glutamine, Serine, and Threonine-rich domain (exon 34). C, qRT-PCR analysis was performed using cDNA from ICN positive (JE131, DO11, TU5) and ICN negative (D510) Ikaros-deficient leukemia cell lines. ND = Not Detected. Y-axis shows target gene expression normalized to expression of the housekeeping gene, HPRT, in each sample. Values were determined by the Pfaffl method and are shown as ratios ((E_target) ^{−CT target}:(E_reference) ^{−CT reference}). E = efficiency of PCR; target = gene of interest; reference = HPRT. Experiments were performed at least twice in duplicate. Error bars indicate SEM.