Previous studies claim that inhibition of the PI3K AKT pathway is in it self adequate to induce apoptosis in neurons. Consequently we investigated whether cell death induced by AKT inactivation was mediated by Puma. To handle this we examined Puma term in CGNs treated with the PI3K inhibitor LY294002 under high potassium supplier Crizotinib circumstances. PI3K inhibition by LY294002 led to a considerable decrease in G AKT levels and a corresponding escalation in Puma protein and mRNA levels. We found that the increase in Puma mRNA expression induced by LY294002 was attenuated in CGNs revealing CA AKT suggesting that AKT inactivation is largely in charge of the LY294002 induced Puma expression. Finally, to ascertain whether Puma is important for neuronal cell death induced by PI3K AKT inactivation we analyzed LY294002 induced apoptosis in CGNs Digestion based on Puma deficient mice and wild type littermates. LY294002 induced significant levels of apoptosis in wild-type but not Puma deficient neurons indicating that Puma is essential for cell death induced by PI3K AKT inactivation, as indicated in Figure 6C. Taken together these results claim that AKT inactivation is just a crucial determinant of Puma induction in neuronal apoptosis. T Glycogen synthase kinase 3b is found to play a pro apoptotic position in several models of neuronal apoptosis including potassium withdrawal in CGNs. GSK3b activity is known to be inhibited by AKT mediated serine 9 phosphorylation and inactivation of AKT leads to activation associated with serine 9 dephosphorylation. Indeed we find that GSK3b serine 9 phosphorylation is decreased in potassium deprived neurons in keeping with its activation, and that IGF 1 prevents this dephosphorylation/ activation.. Similarly, we realize that direct inhibition of PI3K/AKT by LY294002 is enough to induce GSK3b dephosphorylation/ activation.. Consequently, we investigated supplier Cyclopamine whether GSK3b initial may possibly link AKT inactivation to Puma induction and neuronal cell death AKT inactivation may be linked by GSK3b activation. To handle this we examined Puma term in CGNs deprived of potassium in the existence of the GSK3a/b inhibitor SB415286 or even the GSK3b selective inhibitor AR A014418. As demonstrated in Figures 7A and 7B, the induction of protein and Puma mRNA by potassium deprivation was notably paid down by the inhibitors. GSK3b inhibition also significantly paid down the degree of apoptosis induced by potassium deprivation. We next examined the role of GSK3b in Puma expression and cell death caused by LY294002 mediated PI3K/AKT inactivation. Inhibition of GSK3b from the SB415286 element eliminated LY294002 induced Puma mRNA and protein together with LY induced apoptosis. Taken together these results suggest that AKT inactivation triggers Puma induction and neuronal apoptosis using a GSK3b dependent process. W Having established a dependence on both AKT/ GSK3b and JNK pathways in Puma induction we next examined whether these signaling pathways were co dependent or signaling independently of the other person.