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Fig. 11. Notch signaling influences ABCG2 expression directly. (A-D) Immunocytochemical analysis of E18 neurospheres showed that immunoreactivities corresponding to ABCG2 (arrow) and Notch1 (arrowhead) were co-localized in retinal progenitors. Examination of ABCG2 promoters revealed the presence four CSL binding sites (TGGGA) between –1285 and –657 (E). To determine the CSL-mediated effect on the activities of ABCG2 promoter, ABCG2 promoter deletion constructs were transfected into 293T cells, transduced with NICD retrovirus. (F) There was a significant increase in reporter activities with constructs containing CSL sites (b), compared to those without (a), which displayed base level reporter activities. (G) Mobility shift assay carried out with labeled wild-type ABCG2 promoter sequences containing one of the CSL sites (sequence) using HEK293T cell nuclear extracts revealed the formation of complexes, which were similar to those formed with in vitro translated suppressor of hairless (SuH) protein. These complexes could be competed with an excess of unlabeled sequences and were not formed with sequences in which the CSL site was mutated. (H) Nucleosomal DNA immunoprecipitated with CSL antibody in a ChIP assay on E18 retinal progenitors contained sequences corresponding to the Abcg2 promoter, suggesting the presence of CSL on Abcg2 regulatory complexes in vivo.
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