FIG. 3.
Verifying the formaldehyde cross-linking assay. (A and B) Verification of the specificity of the assay using two sets of RKO cells treated with ADR for the times indicated. At the time of harvest, one set was treated with formaldehyde (X-Link) and processed as described in Materials and Methods. The other set was not treated with formaldehyde and processed identically. Immunocomplexes from the extracts were generated using an antibody specific for the pRb protein (A) or the p53 protein (B). PCRs were performed with primers specific for the p21Waf1 promoter (A) or the p53 gene (B). PCR-amplified DNA was resolved by PAGE, and the gels were stained with ethidium bromide. + indicates an amplification performed using genomic DNA as a template; -indicates an amplification performed without genomic DNA. (C) Further analysis to verify specificity. RKO (wt p53-containing) and H1299 (p53-null) cells were treated with ADR for the indicated times. At the time of harvest, the cells were treated with formaldehyde (X-Link) and processed as described in Materials and Methods using PAb1801. The PCRs were performed with primers specific for the p21Waf1 promoter.