text: Preparation of nuclear extracts and electrophoretic mobility shift assays (EMSA)
  Nuclear extracts of purified PBMCs were prepared according to a technique described by Trede et al. [27] and modified for our experimental setup [56]. The intensity of shifted bands was normalized to bands of unstimulated controls. Competition experiments with labeled and unlabeled mutated oligos of the cytokine promoter binding sites (see above) were carried out in order to prevent non-specific binding to nuclear proteins. For such experiments, an excess (5-50x) of unlabeled oligos was added to the nuclear extracts. For specificity of binding, supershift assays for NFkappaB with a combined p65/50 antibody were performed (data not shown). To exclude non-specific reactions, a 30-fold molar excess of Oct-1 DNA (unrelated DNA, Table 1) was used, which did not compete with specific binding. 
  