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Figure 5 | BMC Cancer

Figure 5

From: Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

Figure 5

Specific binding of p53 to IGFBP-3 promoter and its inhibition by methylation. 2 ng of end-labelled 278 bp of Mlu I-Bgl II fragment of pGL-210 were used for EMSA. To construct the methylated probe, a labelled fragment was methylated in vitro with Sss I methylase. Each reaction contained the components listed in Materials and Methods. 2.5 μg of H2O2-treated MCF7 with an induced p53 expression was used as nuclear extract. Supershift was obtained using 0.1 μg of anti-p53 (Ab-2). As competitor, 200 ng of the fragment that carries the core p53 binding site (-210/-149) were used. The mixture was incubated at room temperature for 20 min and analyzed on 4 % nondenaturating polyacrylamide gel in 0.5 × Tris-Glycine buffer. Supershift bands observed in lane 4 were indicated by arrows.

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