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Figure 1 | Molecular Cancer

Figure 1

From: Regulation of MCP-1 chemokine transcription by p53

Figure 1

MCP-1 expression in HPV16 immortalized human keratinocytes and in vitro binding activity of wild-type p53 at the MCP-1 regulatory region. (A) Upper panel: RT-PCR for MCP-1 and GAPDH in HPV16 E6-, E7-, and E6/7 immortalized human foreskin keratinocytes. Cells were treated with TNF-α (250 U/ml) (+) for 6 h. (-) untreated control. The RT-PCR products were separated on 1.5% agarose gel. Lower panel: Western blot analysis of p53. Cytosolic extracts (50 μg per lane) were separated in a 12% SDS-PAGE. Equal loading was confirmed using an actin specific antibody. (B) RT-PCR for MCP-1, p53 and GAPDH in E7-immortalized keratinocytes after lentiviral p53 shRNA delivery. Cells were treated as described in panel A. (C) Schematic overview of the MCP-1 gene. The region -2750 to -2150 bp upstream of the MCP-1 start site harbors two NFκB and one AP-1 binding site. The in silico identified putative p53 binding site (-2422/-2391) relative to NFκB and AP-1 is indicated. Black boxes: exon I-III; white box: polyadenylation site (poly-A), (striped grey box): 3'-regulatory region. (D) Left panel: Electrophoretic mobility shift assay (EMSA): 60 ng of recombinant p53 protein was incubated with [32P]-labeled double-stranded oligonucleotides harboring the wild-type p53-specific RGC-site, the mutated version thereof and the putative MCP-1 p53 binding site, respectively. In addition, binding was also performed in the presence of 200 ng of BSA, anti-GAPDH, DO-1, PAb1801, or PAb421. The positions of free probe, DNA in complex with p53, and DNA in complex with p53 plus supershifting antibodies are indicated by arrows. (D) Right panel: EMSA: comparative binding analysis for wild-type p53 (wtp53) using specific RGC and non-specific RGC mutated versus MCP-1 DNA. DNA binding activities of wild-type p53 (wtp53), p53pro248 and p53pro273 mutants are indicated.

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