U0126 reduces LPS-induced nuclear NF-κB and C/EBPβ binding to the G-CSF promoter in Raw264.7 macrophages.

Abstract

<p>Raw264.7 cells were incubated with DMSO or U0126 (10 μM) for 30 min, then with LPS (100 ng/ml) or PBS for 6 h, then the following tests were carried out. (<b>A</b>) Nuclear levels of p50, p65, C/EBPβ, and lamin B were measured by Western blotting assay. (<b>B</b>) Bindings of p50, p65, or C/EBPβ to the G-CSF promoter were analyzed by ChIP assay via precipitation with antibodies against the test protein or histone H3, used as the control, removal of the antibodies with proteinase K digestion for 12 h at 45ºC, and PCR amplification of a 179-bp G-CSF promoter fragment (-248 to -70 bp) or a 274 bp TNF-α promoter fragment (-270 to -4 bp). Ten per cent of the chromatin DNA used for immunoprecipitation was subjected to PCR and is indicated as ‘input’ (bottom row). In A and B, the data are representative of the results from three independent experiments.</p