Figure 5

p53 binds to the enhancer region of MCP-1 in vivo. (A) DNA sequence of the 5'-regulatory region of the MCP-1 gene (-2550 to -2250) indicating MCP-1 specific primer pairs used for the chromatin immunoprecipitation (ChIP) assay by arrows. The p53 binding site is marked with bold letters. (B) 4Bv cells were maintained at 37°C or shifted to 32°C for 5 h and stimulated with TNF-α for additional 5 h. Cells were harvested for chromatin immunoprecipitation (ChIP) assay as described in Materials and Methods. Samples were subjected to immunoprecipitation without antibody (no ab), with p53 antibody or rabbit IgG (IgG); total lysate was used as a control for PCR amplification (input). (B, left panel) p53 binding was tested by using MCP-1 specific primers; (B, right panel) p21 primers were used as a positive control. Untreated control cells (-); cells treated with 250 U/ml of TNF-α for 5 h (+).