Figure 6

Effects of BMS-345541 and wortmannin on LPS signalling in chondrocytes. (A) Effects of BMS-345541 or wortmannin on LPS-induced IκB-α phosphorylation and degradation in chondrocytes in monolayer cultures. Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml lipopolysaccharides (LPS) or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by LPS treatment (100 ng/ml) for the indicated times. Cytoplasmic extracts were prepared, fractionated (500 ng protein per lane) on 10% SDS-PAGE and electrotransferred onto nitrocellulose membranes. Western blot analysis was performed with anti-phospho-specific-IκB-α (II), anti-IκB-α (I) and anti-β-actin (II, control). The results shown are representative of three independent experiments. (B) Effects of BMS-345541 or wortmannin on LPS-induced IKK activation in chondrocytes in monolayer cultures. Primary human chondrocytes in monolayer culture were either stimulated with 100 ng/ml LPS or prestimulated with 5 mM BMS-345541 or with 20 nM wortmannin for 12 h followed by treatment with 100 ng/ml LPS for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IkB kinase (IKK) and then analyzed by an immune complex kinase assay as described in Materials and methods. To examine the effect of LPS, BMS-345541 or wortmannin on the level of activation of IKK proteins, whole-cell extracts were fractionated (500 ng protein per lane) on SDS-PAGE and examined by western blot analysis using anti-phospho-specific-IκB-α and anti-IκB-β (I). LPS, BMS-345541 or wortmannin had no direct effect on the expression of IKK-α or IKK-β proteins (II, III). The results shown are representative of three independent experiments.