A–D: Effects of IGF-1 or/and PDGF-bb or IKK-inhibitor (BMS) on the IL-1β-induced IKK activation in chondrocytes in monolayer cultures.

<p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb, a combination of both growth factors (5 ng/ml each) or 5 µM BMS-345541 (BMS) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IκB kinase (IKK)and then analyzed by an immune complex kinase assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028663#s2" target="_blank">Materials and Methods</a>. To examine the effect of <i>IGF-1 or/and PDGF-bb or</i> BMS 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-IKK-α and anti-IKK-β antibodies. The results shown are representative of three independent experiments.