Figure 8.

ChIP assay shows the mutant p53–mediated increase in transcription factor binding on the Axl promoter. ChIP analysis was performed on H1299 cells stably transfected with a vector (HC5) or mutant p53-R273H (R273H) as described for Figure 6 to test whether transcription factors are preferentially binding to the Axl promoter. Cells were cross-linked with formaldehyde, harvested, and DNA sonicated to generate 500-bp to 2-kb fragments. Designated proteins were immunoprecipitated using respective antibodies. Cross-linking was reversed and the DNA isolated. qPCR was performed using gene-specific primers. Normalized values represent qPCR values normalized to a fragment 5 kb upstream of the Axl gene not affected by mutant p53. ChIP was performed with antibodies directed against CREB, E2F1, and p300. Experiments were performed in triplicate. Error bars showing standard deviations are indicated.