Pharmacological inhibition of p38 or PC-PLC and its effect on induced activation of IKKβ and NF-κβ DNA-binding affinity.
(A-D) HUVECs were preincubated for 30 minutes with increasing concentrations of SB202190 (1.25, 5, or 20 μM) (A, C) or D609 (1.56, 6.25, or 25 μg/mL) (B, D) and subsequently either left untreated or stimulated for 1 hour with 1.5 mM NiCl2 or 2 ng/mL TNFα as indicated. Cell lysates were subsequently assayed for IKKβ activity. (E) HUVECs were pre-incubated with 1.25 (lane 3), 5 (lane 4), or 20 μM SB202190 (lane 5) or with 1.56 (lane 6), 6.25 (lane 7), or 25 μg/mL D609 (lane 8) and subsequently stimulated with NiCl2 or TNFα as indicated. Thereafter, nuclear extracts were obtained and an electrophoretic mobility shift assay (EMSA) was performed as described in “Materials and methods.” For lanes 13 through 18, 20 μM SB202190 or 25 μg/mL D609 was added to nuclear extracts only during the EMSA reaction procedure in order to study potential interaction of inhibitors with the binding of NF-κB to its cognate binding site. I and II indicate the major NF-κB complexes formed as determined by competition assays with unlabeled probes (lanes 19 and 20).
