Figure 5

LPS-induced NF-κB and NADPH activation in BMDMs is blocked by PHA 568487. (A) Cultured BMDMs were stimulated with LPS (100 ng/mL) for 2 h and immunostained for nuclear phosphorylated NF-κB subunit p65. Preincubation with PHA 568487 (10 µg/mL) for 30 min significantly reduced activation and nuclear translocation of p65 (LPS+PHA). NF-κB activation was undetectable in unexposed cells (medium) or in cells exposed to PHA 568487 (PHA). (B) NADPH oxidase mediated superoxide generation from BMDMs was measured by chemiluminescence method. LPS stimulation at 10 ng/mL for 24 h significantly increased superoxide generation from BMDMs (***P < 0.001 versus C), which was abolished by preincubation of PHA (^&&&p< 0.001 versus LPS). PHA perse did not significantly affect superoxide generation in nonstimulated BMDMs. Data are expressed as mean ± SEM and compared by one-way ANOVA and Bonferroni posthoc, n = 6. C, control; PHA, PHA 568487