Figure 3

Proteolytic cleavage and activation of protein kinase Cδ (PKCδ) downstream of tumor necrosis factor receptor 1 (TNFR1) in N27 dopaminergic cells. (A) TNFR1-dependent proteolytic activation of PKCδ. N27 cells were treated with tumor necrosis factor (TNF) (30 ng/ml) for 6 h or pretreated with either a TNFR1 neutralizing antibody (TNFR1-nAb, 20 μg/ml) or etanercept (5 μg/ml) for 30 minutes and lysates were probed for PKCδ proteolytic cleavage by Western blotting. TNF induced strong proteolytic cleavage of PKCδ that could be blocked by neutralizing TNFR1 receptor signaling. (B) Band intensities for cleaved PKCδ were quantified using densitometric analysis and normalized to β-actin levels. (C) Immunoprecipitation (IP)-kinase assay for PKCδ activation. IP-kinase assays were performed on the same cell lysates used for Western blots. PKCδ was immunoprecipitated from 500 μg of total protein from each sample and used for the in vitro kinase activity assay with a histone substrate and radiolabeled [γ-³²P] ATP. PKCδ activity was quantified using densitometric analysis of the ³²P-histone band intensity and expressed as percent control. The proteolytic cleavage of PKCδ induced by TNF treatment was accompanied by a concomitant increase in kinase activity and was dependent on signaling through the TNFR1 receptor. A representative kinase assay gel is shown. Data represent the group mean ± SEM of densitometric values obtained from three independent experiments. *** (P <0.001) denotes significant differences compared to control cells and ## (P <0.01) denotes significant differences compared to TNF-treated cells.