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. 2009 Jul 31;284(41):27933–27943. doi: 10.1074/jbc.M109.025742

FIGURE 5.

FIGURE 5.

p65 binds the authentic PDGF-Rα promoter in cells exposed to IL-1β. A, NIH-3T3 cells were incubated with IL-1β for 1 h prior to lysis and ChIP analysis (upper). The veracity of the p50 antibody (Ab) as a ChIP reagent was determined by its ability to pull down the human VCAM-1 promoter in IL-1β-treated extracts (lower). TI, total input. No antibody added (No Ab) represents a negative control. B, EMSA using 32P-oligonucleotide PDGF-Rα−541/−511 and nuclear extracts (NE) of cells incubated for 1 h with IL-1β. Where indicated, nuclear extracts were incubated with indicated antibodies prior to the addition of the probe. The arrow denotes inducible nucleoprotein complex. C, EMSA with 32P-oligonucleotide PDGF-Rα−541/−511 or 32P-oligonucleotide PDGF-Rα−541/−511 and recombinant 100 ng of p65/p65. The sequence of oligonucleotide PDGF-Rα−541/−511 is 5′-GCA ACT ACA CGG CAC TTT CCC TTA AGA CCC-3′, and sequence of mutated oligonucleotide PDGF-Rα−541/−511 is 5′-GAC ACT ACA CAA TAC TTT TTT TTA AGA CCC-3′ (NF-κB motif underlined, mutations bolded).