Figure 2

Identification of Ets binding motifs essential for CD133 P5 activity. A, Deletion analysis of the promoter activity of CD133 P5. Left panel, schematic representation of luciferase reporter constructs containing the deleted sequence of P5 region. +1 indicates the transcription start position of exon1E1. Right panel, luciferase activities of Caco-2 and Fuji cells transfected with a series of deletion mutants. Data are means ± s.d. of values from three independent experiments. *P < 0.05. B, Effects of mutation of Ets binding sites in the -98/-25 region of the CD133 P5 on the transcriptional activity. Left panel, schematic representation of P5-98-based luciferase reporter constructs containing point mutation in two Ets binding sites (EBS#1 and EBS#2). Open circles represent wild-type sequence (GGAA) and closed circles represent substituted sequence (TTAA). Right panel, luciferase activities of Caco-2 and Fuji cells transfected with the indicated mutant plasmids. *P < 0.05, **P < 0.01. C, EMSA analysis of nuclear proteins binding to oligonucleotides containing the Ets binding site of the CD133 P5. The arrowheads a to d indicate specific complexes of nuclear proteins with GGAA sequence in EBS#2. N.S.: non-specific binding of proteins.