FIGURE 4.

The p53 protein binds the essential CerS6 promoter. A, schematic depicting pulldown of CerS6 promoter fragment bound to p53 by immunoprecipitation with p53-specific antibody (Ab). The fragment corresponding to 264-bp CerS6 promoter was excised from pGL3/CerS6 vector, incubated with purified recombinant p53, cross-linked with formaldehyde, and pulled down using p53-specific antibody/protein G beads. After the pulldown, cross-linking was reversed by 5 m NaCl. The pulled down sequence was amplified by PCR using fluorescently labeled sequence-specific primers (shown in Table 1). B, agarose gel electrophoresis of the amplified CerS6 fragment after the pulldown (as depicted by panel A). Lane 1, pulldown of the CerS6 promoter using recombinant p53 and p53-specific antibody; lane 2, pulldown with ALDH1L1 protein and ALDH1L1-specific antibody; lane 3, pulldown with human IgG; lane 4, same as lane 1 but without cross-linking. The gel was stained with ethidium bromide, and the image was obtained using an Odyssey Fc instrument (LI-CORE). The green channel was assigned to image the ethidium bromide-stained double-stranded DNA; the red channel imaged the fluorescent primer. Double-stranded DNA containing the fluorescent label was seen in yellow due to the two-channel overlay.