FIGURE 7.

TNFα activates PKCϵ and NF-κB via PC-PLC. LNCaP cells were incubated for 30 min with either the PI-PLC inhibitor U73122 (30 μm) or the PC-PLC inhibitor D609 (50 μm) and then stimulated with TNFα (10 ng/ml) or vehicle. A, cytosolic and particulate fractions were prepared by ultracentrifugation 30 min after TNFα treatment. Endogenous PKCϵ levels were determined in each fraction by Western blot analysis. B, effect of PI-PLC and PC-PLC inhibitors on GFP-PKCϵ membrane translocation by TNFα (50 ng/ml), as determined by real-time microscopy. C, NF-κB DNA binding activity was evaluated in nuclear extracts by EMSA 30 min after stimulation with TNFα. Relative optical density is indicated underneath each lane. NC, negative control, no protein added. D, LNCaP cells were infected with either PKCϵ or control (LacZ) AdVs (multiplicity of infection = 1 pfu/cell) and, 24 h later, treated with either TNFα (10 ng/ml) or vehicle for 30 min. Phosphorylated and total IκBα levels were determined by Western blot analysis. In all cases, similar results were observed at least in three independent experiments.