We next examined p JNK localization by immunostaining to determine the subcellular distribution of p JNK protein, to better comprehend the mechanism of JNK activation induced by NGF withdrawal. Under normal culture conditions, DRG neurons showed punctate ALK inhibitor JNK to g staining throughout the cell human body and neuronal processes in both wt and DLK neurons. Curiously, NGF starvation resulted in a re-distribution of p JNK from axons to cell bodies over a period of 4 h, which did not occur in DLK neurons. Staining of countries using an antibody directed to Tuj1 established that the absence of p JNK labeling in axons was not an effect of the degenerating but rather a specific relocalization of p JNK to the cell body. The moment of p JNK relocalization strongly correlated with how many neurons that stained beneficial locomotor system for p c Jun, consistent with the hypothesis that nuclear localization of p JNK is needed for c Jun phosphorylation and neuronal apoptosis. For that reason of NGF withdrawal to determine the functional part of the increased JNK activity observed in DRG neurons, we tested the effect of JNK inhibitors on NGF withdrawal induced degeneration. Pharmacological inhibition of JNK activity was sufficient to significantly reduce degrees of caspase 3 activation observed in dissociated DRG cultures and recovery axons from degeneration induced by NGF deprivation. These protective effects were much like those noticed in DLK neurons. As small molecule inhibitors could prevent numerous kinases in addition to their preferred goal, this test was repeated with two additional structurally unique JNK inhibitors, which gave similar results. These data support a system in which DLK is required for activation of the JNK d Jun stress response pathway that occurs in neurons consequently of NGF deprivation, and this JNK activity results in neuronal apoptosis and destruction of axons. The statement that DLK neurons retain purchase Celecoxib regular localization and levels of p JNK when cultured in the presence of NGF, yet show deficiencies in p JNK relocalization and attenuated phosphorylation of c Jun in NGF starvation paradigms, suggested that DLK is able to selectively modulate the prodegenerative areas of JNK signaling. We hypothesized that this might be achieved through the discussion of DLK using a specific JIP to make a signaling complex that will allow for restricted JNK activation. To check this possibility, we examined whether siRNA based knock-down of individual JIPs could phenocopy the protective effects observed in DLK neurons. Interestingly, siRNA based knockdown of JIP3 provided similar degrees of protection to those observed after knockdown or knock-out of DLK, while negligible protection was provided by JIP1 siRNAs despite productive knockdown of JIP1 protein. We tested whether both of these proteins interact when coexpressed in HEK 293 cells, to determine whether JIP3 and DLK can develop a signaling complex. Immunoprecipitation of Flag described DLK could pull-down coexpressed Myctagged JIP3 but not a GFP control, indicating that these proteins can interact.