7 research outputs found

    A–B: Effects of Src-, PI-3K-, AKT-inhibitors and IGF-1 or/and PDGF-bb on IL-1β-stimulated phosphorylation of NF-κB in chondrocytes.

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    <p>Primary chondrocytes were either stimulated with 10 ng/ml IL-1β, pre-stimulated with PP1 (10 µM), wortmannin (20 nM) and SH-5 (10 µM) for 1 h (A), or with 10 ng/ml IGF-1, 10 ng/ml PDGF-bb or a combination of both growth factors (5 ng/ml each) for 12 h (B) and then incubated with IL-1β for 30 min. Nuclear extracts were subjected to 10% SDS-PAGE (500 ng protein per lane), transferred to nitrocellulose membranes and then probed using an antiserum reactive with an anti-phospho-p65 or anti-PARP polyclonal antibody (housekeeping control). Similar results were obtained in three independent experiments.</p

    A–C: Effects of IGF-1 or/and PDGF-bb on the IL-1β-induced p65 acetylation in chondrocytes in monolayer cultures.

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    <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 or a combination of both growth factors (5 ng/ml each) for 12 h and treated with 10 ng/ml IL-1β for the indicated times. Whole-cell extracts were prepared and immunoprecipitated with an anti-p65 antibody. Western blot analysis was then performed with an anti-acetyl-lysine antibody or with an anti-p65 antibody. The results shown are representative of three independent experiments.</p

    Effects of IGF-1 or/and PDGF-bb on IL-1β-induced inhibition of cartilage ECM expression in chondrocytes.

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    <p>Western blot analysis was performed to evaluate the effects of <i>IGF-1 or/and PDGF-bb</i> on IL-1β-induced inhibition of chondrogenic potential in chondrocytes. Whole cell lysates were probed with antibodies to collagen type II, cartilage specific proteoglycan (CSPG) and β1-integrin. Primary chondrocytes were treated with 10 ng/ml IL-1β, 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or 5 ng/ml PDGF-bb and 5 ng/ml IGF-1, or were pre-treated with 10 ng/ml PDGF-bb, 10 ng/ml IGF-1 or 5 ng/ml PDGF-bb and 5 ng/ml IGF-1 for 12 h and then stimulated with IL-1β for 24. Untreated chondrocytes had strong production of collagen type II, CSPG and β1-integrin, and stimulation with IL-1β alone markedly reduced these proteins. Pre-treatment of the chondrocytes with IGF-1 or/and PDGF-bb, however inhibited adverse effects of IL-1β. This was confirmed by quantitative densitometry. Expression of the housekeeping gene β-actin remained un-affected.</p
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