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Figure 1 Assessment of Spontaneous NF-B Activation in Circulating Monocytes (Protocol A)

(A) Electrophoresis mobility shift assay (EMSA). In each patient, freshly isolated peripheral blood monocytes (5 x 10⁵) were analyzed for the spontaneous presence of activated nuclear factor (NF)-B complexes in their nuclei by EMSA in the presence of anti-p50 subunit monoclonal antibodies or isotype-matched control IgG antibodies. As a positive control (Pos) nuclear extracts were generated from 5 x 10⁵ monocytes incubated for 30 min with 10 ng/ml lipopolysaccharide (LPS), and as a negative control (Neg) nuclear extracts were generated from 5 x 10⁵ monocytes incubated for 30 min with phosphate-buffered solution only. NF-B heterodimers containing the p50 subunit were detected in 82% of group 1 patients, 14% of group 2 patients, and 21% of group 3 patients (p < 0.001; group 1 vs. group 2 and group 3). The figure shows the assessment of NF-B activation in 3 patients (Pt) of group 1 and in 3 patients of group 2. (B) Enzyme-linked immunosorbent assay (ELISA) for active NF-B p65. The amount of activated NF-B p65 subunit in nuclear extract (5 µg), prepared from 5 x 10⁵ monocytes was assessed by a sensitive ELISA assay for active NF-B. The activated NF-B p65 subunit was significantly higher in group 1 than in the other groups. ANOVA = analysis of variance; OD = optical density.