Effect of PD98059 on LPS-induced nuclear binding of NF-κB and AP-1 and κB- and AP-1–dependent transcription in THP-1 cells.

(A) Nuclear extracts were prepared from THP-1 cells preincubated with or without PD98059 (50 μM) for 30 minutes before stimulation with LPS (10 μg/mL) for 2 hours. EMSAs were used to measure binding of p50/p65 and c-Rel/p65 to oligonucleotides containing κB sites from the murine IgκB and human TF genes. Two nonspecific (ns) complexes were observed binding to the IgκB oligonucleotide. (B) The p(κB)₄-LUC (3 μg) was transfected into THP-1 cells. After transfection, cells were preincubated in the presence or absence of PD98059 (50 μM) for 30 minutes before incubation with or without LPS for 5 hours at 37°C. (C) The p(κB)₄-LUC was cotransfected with either vector control (pcDNA3) or a plasmid expressing a dominant-negative ERK1 mutant. (D) EMSAs were used to measure c-Fos/c-Jun binding to an AP-1 site using nuclear extracts from cells stimulated with LPS (1 hour) with or without PD98059. (E) LPS-induced, AP-1–dependent transcription was measured in cells transfected with p(AP-1)₄-LUC in the presence and absence of PD98059. (F) LPS-induced, AP-1–dependent transcription was measured in the presence and absence of a plasmid expressing a dominant-negative ERK1 mutant. Transfection data are shown from 6 independent transfection experiments and are expressed as mean ± SD.