Figure 6.

Mechanism of TNFα regulation by the ACE C-domain. A, measurement of superoxide generation by WT and transgenic macrophages (Tg-ACE, Tg-NKO, and Tg-CKO). Peritoneal macrophages were isolated and challenged with 10 μm PMA. After 10 min, superoxide generation was measured by cytochrome c reduction. B, to study the role of ROS in macrophage polarization, macrophages were treated with B16-F10 supernatant for 24 h in the presence of DPI, and then the production of TNFα, phospho-NF-κB p65, and phospho-STAT1 was measured by ELISA (n = 4). C, measurement of C/EBPβ by Western blotting. D, measurement of IL-4Rα by Western blotting. E, for blocking IL-4Rα in macrophages, the cells were conditioned with melanoma supernatant for 24 h in the presence of IL-4Rα neutralizing antibody (500 ng/ml). TNFα was then measured by ELISA.