3.

IL-1β enhances Sp1/Sp3 binding to TβRII promoter (position −25). A: Electrophoretic mobility shift assay (EMSA) reactions were performed using the 5′ end-labelled −47/−15 probe, incubated with 6 μg of nuclear extract from chondrocytes treated in the absence or in the presence of IL-1β for 1 or 3 hrs. EMSA reactions were also performed with 3-hrs IL-1β sample in the presence of 100-fold molar excess of the wild-type, mutated −47/−15 unlabelled probe or Sp1, NFκB and AP1 consensus sequences. Untreated samples were also pre-incubated with Sp1, Sp3 and p65 antibodies (Ab) before incubation with the probe. B: Nuclear extracts were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-Sp1 or anti-Sp3 antibodies. C: After 24 hrs of incubation with IL-1β, cells were fixed with formaldehyde, lysed and underwent digestion. in vivo cross-linked chromatin was then precipitated independently using Sp1, Sp3, p65 antibodies or IgG. The recovered immunoprecipitated DNA was then used for PCR with specific primers for the TβRII promoter segment of interest. The amplicons were analysed by electrophoresis on a 3% agarose gel. Sp1, Sp3 and p65 relative binding of three independent experiments are shown in histograms.