Figure 3

Characterization of the transcription factors responsible for Sp1 binding sites in the c1orf109 gene promoter. (A) Distribution of DNA fragments by sonication. DNA fragments were sheared with a distribution of fragments from 200 to1000 bp. (B) Chromatin immunoprecipitation assay using HeLa cell lysates. Chromatin was immunoprecipitated using antibody against Sp1. Lanes 1, 5 and 9 were PCR products from immunoprecipitation using primers ChIP 1 F/R, ChIP 2 F/R and ChIP 3 F/R respectively. Lanes 2, 6 and 10 were PCR products from WCE using primers ChIP 1 F/R, ChIP 2 F/R and ChIP 3 F/R respectively. Lane 3, 7 and 11 were PCR products using ChIP InpF/R from WCE. Lanes 4, 8 and 12 were PCR products using ChIP InpF/R from immunoprecipitation. WCE, whole cell extract. (C) Biotin labeled oligonucleotides corresponding to GC boxes were used in an EMSA with nuclear proteins from HeLa cells. Specific shift-bands are indicated with closed arrowheads. NE, nuclear extract; SC, 100× specific competitors; NSC, 100× nonspecific competitors.