Figure 4

TMP does not impair NFκB binding in vitro to the TNF-α promoter. (A) Nuclear extracts from untreated (lane 1) RAW 264.7 cells or cells stimulated 4 hrs with 1 μg/ml LPS (lanes 2-6) were incubated with a radiolabeled ds oligonucleotide probe to the κB3 site of the TNF-α promoter. Probes were incubated with nuclear extract alone (lanes 1 and 2), in the presence of 100-fold molar excess of unlabeled wt (lane 3) or mutant κB3 competitor oligonucleotides (lane 4), or in the presence of the indicated Abs (lanes 5 and 6). Specific nucleoprotein (filled arrows) and Ab-supershifted complexes (empty arrows) are indicated. (B) The impact of TMP on protein binding to TNF-α κB3 (upper panels) or control NF-Y (bottom panel) was assessed in nuclear extracts from LPS-treated RAW 264.7 cells co-stimulated with TMP (lane 3) or upon addition of exogenous TMP to the binding reaction (lanes 4-6, 0.25 μM, 2.5 μM, and 25 μM, respectively).