Figure 6.

Modulation of PKC-mediated regulation of NET activity is dependent on long term processes. A destabilized version of MKP3 (MKP3-DD) was stably expressed in PC12 cells. Short term stabilization of this destabilized MKP3 was achieved pharmacologically by a 4-h incubation with the Shield1 compound (A). Following restoration of the MKP3 protein by Shield1 (1 μM for 4 h) PKC was activated by PMA for 30 min and NET-specific radiolabeled uptake was performed as above comparing with vehicle treated control (B). All experiments were performed in triplicate in three independent experiments. No significant attenuation of PKC-mediated down regulation of NET was observed following MKP3 protein restoration (two-way ANOVA with Bonferroni post hoc analysis). A dominant negative mutant of the ras protein (RasN17) was stably expressed in PC12 cells and PKC was activated by PMA for 30 min and NET-specific radiolabeled uptake was performed as above comparing with vehicle treated control (C). All experiments were performed in triplicate in three independent experiments. PKC mediated down regulation was completely inhibited as there was no significant difference between vehicle and PMA treated cells (Student’s t-test).