Figure 2

Tumor necrosis factor (TNF) induced proteolytic cleavage of protein kinase Cδ (PKCδ) in dopaminergic N27 cells. (A) Time-dependent PKCδ proteolytic activation. N27 dopaminergic cells were treated with recombinant TNF (30 ng/ml) for 3 h and 6 h and lysates were probed by Western blotting with an antibody to the C-terminus that detects both the native protein (78 kDa) and the proteolytically cleaved catalytic fragment (38 kDa). TNF treatment caused a time-dependent increase in PKCδ cleavage, which peaked at 6 h. (B) Band intensities for cleaved PKCδ from three blots quantified using densitometric analysis and normalized to β-actin. (C) Dose-dependent proteolytic activation of PKCδ by TNF treatment. N27 cells were treated with increasing doses of TNF (0 to 60 ng/ml) for 6 h and lysates were probed for PKCδ by Western blotting. TNF induced a dose dependent increase in PKCδ cleavage, starting at 10 ng/ml. (D) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. (E) Caspase-3 and caspase-8 dependent proteolytic cleavage of PKCδ. N27 cells were treated with 30 ng/ml of TNF alone for 6 h or in the presence of peptide inhibitors (25 μM) of caspase-8 (Ac-IETD-fmk) and caspase-3 (Ac-DEVD-fmk). Lysates were probed for PKCδ proteolytic cleavage by Western blotting. Proteolytic activation of PKCδ by TNF treatment was reduced by inhibition of caspase-3 and caspase-8. (F) Band intensities for cleaved PKCδ from three blots were quantified using densitometric analysis and normalized to β-actin. Data is expressed as a fold change over control. * (P <0.05) and ** indicate significant differences compared to control cells; # (P <0.05) indicates significant differences compared to TNF-treated cells.