The PI3K/AKT pathway mediates AC-induced inhibition of NF-κB activation in NOD DCs. NOD or NOD.MerTK^KD BMDCs (A-C,E,G) and sDCs (D,F) were incubated with 200 nM Wort or 50 μM LY for 1 hour and then treated with ACs for 3 hours or left untreated. In some experiments (C-G), DCs were subsequently stimulated with LPS (50 ng/mL) for 0.5 hours. (A) In vitro AKT activity was determined by measuring phosphorylation of a GSK3 substrate by Western blot. The same blot was reprobed for AKT protein. (B) NOD BMDCs were pretreated either with isotype control or αMerTK Ab for 1 hour and then incubated with ACs at specified times. Phosphorylation of AKT in cytoplasmic extracts was determined via Western blot using an αphospho AKT Ab. The same blot was reprobed for AKT protein. (C-D) Nuclear NF-κB or OCT-1 DNA binding activity was determined via EMSA. (E-F) Cytoplasmic IκBs and β-actin protein were detected by Western blot using the same blot. (G) In vitro IKK activity was measured as in Figure 2. The same blot was reprobed for IKK1 and IKK2 protein. Data are representative of 3 independent experiments.