Figure 4.

Cross-linking of Sp1 and p53 after DLL4 Notch1 activation and dislocation of Sp1 from EGFR promoter assessed by ChIP. (A) Cross-linking assay with DMP agent was performed in unstimulated and DLL4-stimulated H1975 cells, and whole cell lysates were analyzed by western blot to detect the free forms of Sp1 and p53. Vinculin was used to ensure equal loading. (B) ChIP assay was performed by chromatin immunoprecipitation with an anti-Sp1 antibody in unstimulated and DLL4-stimulated H1975 cells. Immunoprecipitated DNA fragments were then amplified using EGFR promoter specific primers set 4. Histone H3 was used as positive control. Levels of amplified products were analyzed on a 2% agarose gel. (C) Quantitative analysis of ChIP assay was performed by RT-PCR. Data were expressed as % of input in unstimulated and DLL4-stimulated H1975 cells. At least three independent experiments were performed and data are expressed as mean ± SE. The symbol *, p < 0.05.