FIG. 2.

E2F4 binding to the DHFR gene is promoter specific and requires an intact E2F binding site. As a demonstration of the specificity of our formaldehyde cross-linking assay, we monitored binding of E2F4 to three regions of the DHFR gene in NIH 3T3 cells (A) and to both the endogenous and integrated DHFR promoter construct in NIH 3T3 NW luc cells (B). Cross-linked chromatin from asynchronously growing cells was incubated with an antibody against E2F4 or in the absence of antibody (No Ab). Immunoprecipitates from each sample were analyzed by PCR using primers specific for different regions of the DHFR promoter. As a positive control, a sample representing 0.03% of the total input chromatin (input) was included in the PCRs. Additional controls include a precipitation lacking both antibody and chromatin (Water).