Influence of TRAF2 on CD40-mediated cytokine secretion in macrophages. Macrophages were obtained by exposure of human blood monocytes for 6 days to M-CSF (100 ng/mL) and then transfected with an empty vector (Co) or a TRAF2-expressing vector (TRAF2). Twenty-four hours after transfection, macrophages were treated with 500 ng/mL CD40L. (A) Immunoblot analysis of TRAF2 expression, 24 hours after transfection. Peripheral blood monocytes were used as a control. (B) (Top panel) Cytokines were detected in the culture supernatant of control (Co) and TRAF2-transfected cells treated with CD40L for 24 hours using an antibody array. (Bottom panel) Spots were quantified by densitometry analysis and reported to internal control (C+). The ratio between cytokine quantities detected in control and TRAF2-transfected macrophage supernatants was quantified. (C-E) ELISA quantification of IL-8 and MCP-1 secretion in (C) control (Co) and TRAF2-transfected cells (TRAF2) treated with CD40L for 24 hours (each line indicates an independent sample) or (D) control (□) and TRAF2-transfected cells (■) treated with CD40L for the indicated times (hours). Results are the mean plus or minus SD of 3 measurements in one sample. (E) Control (Co) and cIAP1 siRNA-transfected cells (2 sequences 1 and 2) treated with CD40L for 24 hours (mean ± SD of 3 independent samples normalized to the control).