1). IKK-β leads to nuclear exclusion and protein degradation of FOXO3 [[16]]. To determine if IKK-ε promotes the same phenomenon, FLAG-tagged expression constructs encoding IKK-β and IKK-ε, as well as their dominant

negative forms, were expressed in the 293-TLR4 cells. As expected, IKK-β expression was associated with reduced FOXO3 nuclear localization, while expression of its dominant negative mutant (IKK-β-KA) had no effect (Fig. 1B). Decreased levels of FOXO3 were also observed in nuclear fraction of the IKK-ε- but not IKK-ε-KA-expressing selleck kinase inhibitor cells, suggesting that similarly to IKK-β, IKK-ε induces nuclear exclusion. In addition, a slow migrating band (indicated by an arrow) detected in cells expressing IKK-ε (Fig. 1B), consistent with direct or indirect IKK-ε-mediated posttranslational modifications of FOXO3, for example Navitoclax phosphorylation. Next, we examined whether IKK-ε can physically interact with FOXO3. HA-tagged FOXO3 protein (HA-FOXO3) was expressed in the 293-TLR4 cells together with FLAG-tagged IKK-β, IKK-ε, or bacterial alkaline phosphatase (BAP) as a negative control, and immunoprecipitated (IP) (Fig. 2A). Consistent with previous findings [[16]], FOXO3 interacted with IKK-β. It also formed complexes with IKK-ε, but not with BAP (Fig. 2A).

To examine if this association was inducible upon TLR4 stimulation, 293-TLR4 cells, which stably express TLR4/MD2-CD14 receptors, were treated with lipopolysaccharide (LPS). IKK-ε/FOXO3 interaction was slightly enhanced by LPS treatment (Fig. 2A), suggesting that FOXO3 recruitment by IKK-ε is potentiated by LPS stimulation. This observation was confirmed in a time course experiment which demonstrates that IKK-ε-FOXO3 complex formation increased as early as 5 min, reached its maximum at 30 min, and returned to the basal level after 120 min post LPS stimulation

(Supporting Information Alectinib mouse Fig. 2A). The rapid and transient kinetics of IKK-ε-FOXO3 complex formation suggests that IKK-ε may signal to FOXO3 in response to TLR4 activation. Next, we examined whether an interaction between the endogenous IKK-ε and FOXO3 could be detected in human monocyte-derived DCs (MDDCs) and if this interaction may be induced by LPS stimulation. FOXO3 was IP and western blot (WB) analysis for IKK-ε revealed a specific interaction with FOXO3, which was induced after LPS stimulation (Fig. 2B). Further mapping of the interaction interface using deletion mutants of HA-FOXO3 revealed that C-terminus of FOXO3 protein is critical for IKK-ε-FOXO3 interaction (Fig. 2C). To determine if slow migrating bands observed in protein extracts of the cells expressing IKK-ε (Fig. 1B, 2A and C), correspond to phosphorylated forms of FOXO3, the extracts were treated with lambda-phosphatase to remove all phosphate groups. After phosphatase treatment, only one band of the right size was detected (Supporting Information Fig. 2B), demonstrating that IKK-ε induces FOXO3 phosphorylation.