Fig. 6

Effects of DEX on phosphorylation and DNA binding of NF-κB p65. a ASMCs were incubated for the indicated time in pH 6.3 or pH 7.4-adjusted medium. As a positive control of serine 536 phosphorylation and DNA binding of NF-κB p65, ASMCs were stimulated with 10 ng/ml TNF-α in the pH 7.4-adjusted medium. A weak increase of phospho-NF-κB p65 (Ser 536) is observed in ASMCs at 30–60 min after pH 6.3-stimulation, but not after pH 7.4-stimulation. GAPDH was used as an endogenous control protein. Representative data of 3 independent experiments are shown. b ASMCs were pretreated with 30 μM BAY11–7082 or control vehicle (0.1% DMSO) for 30 min. The cells were further incubated for 60 min in the pH 6.3-adjusted medium, pH 7.4-adjusted medium, or pH 7.4-adjusted medium containing 10 ng/ml TNF-α. BAY11–7082 almost completely decreases the expressions of phospho-NF-κB p65 (Ser536) in all cases, pH 6.3, pH 7.4, and TNF-α (pH 7.4). Representative data of 3 independent experiments are shown. c After 30-min preincubation with DEX (1 μM) or control vehicle (0.1% ethanol), ASMCs were stimulated for 60 min by pH 6.3-adjusted medium, pH 7.4-adjusted medium, or pH 7.4-adjusted medium containing 10 ng/ml TNF-α. DEX does not affect serine 536 phosphorylation of NF-κB p65 in all cases, pH 6.3, pH 7.4, and TNF-α (pH 7.4). Representative data of 3 independent experiments are shown. d Binding of NF-κB p65 to its consensus DNA sequence is significantly increased at 60 min after pH 6.3- or TNF-α-stimulation compared with pH 7.4-stimulation (P < 0.01). DEX (1 μM) does not substantially affect the binding of NF-κB p65 to its consensus DNA sequence in all cases, pH 6.3, pH 7.4, and TNF-α (pH 7.4). Data are expressed as percentages of absorbance at 450 nm (OD450) of pH 6.3-stimulated ASMCs without DEX (mean ± SEM, n = 5; NS, not significant)