Figure 3: IL-27 increases the expression of p47^phox in macrophages.

(a) M-Mac were transfected with either control si-RNA (si-Ctlr) or si-RNA targeting p47^phox (si-p47) followed by treatment with or without 100 ng/ml IL-27 for 48 h at 37 °C. As a control, un-transfected cells (Mock) were treated with IL-27. 48 h after transfection, whole cell lysates were prepared and western blotting was performed using anti-p47^phox or anti-β-actin antibodies. (b) si-Ctlr, si-p47 or Mock-transfected cells were treated with IL-27 for 48 h at 37 °C and superoxide production was analyzed by measuring H₂O₂ within culture supernatants. Data shown represent means ± SD of triplicate samples. (c) Macrophages were transfected with an empty or a p47^phox expression vector for 6 h, and then the expression levels of p47^phox were determined by Western blot. (d) H₂O₂ induction by PMA stimulation was detected by ROS assays following transfection of either the empty or p47^phox expression vector into macrophages. Data shown represent means ± SD of triplicate samples. (e,f) Monocytes from a healthy control or a CGD patient lacking the expression of p47^phox were differentiated into macrophages. Cells were then incubated with or without IL-27 for 48 h at 37 °C. The resulting cells were then stimulated with or without 100 ng/ml PMA for 30 min and ROS activity was measured by detection of H₂O₂ in culture supernatants. Data shown represent means ± SDs of triplicate samples from three independent studies. *P < 0.01.