Profound failure to inhibit GSK-3 in RAPA-DC promotes increased IL-12p70 secretion after LPS stimulation. The activity of GSK-3α/β in murine CTR- and RAPA-DCs, determined by the extent of phosphorylation of inhibitory N-terminal serines (S21 for GSK-3α; S9 for GSK-3β) versus facilitatory tyrosines (Y279 in GSK-3α; Y216 in GSK-3β), was assessed by Western blot. (A) CTR-DCs demonstrated a profound increase in the inhibitory phosphorylation of S21/9 after TLR4 ligation. However, RAPA-DCs, either unstimulated or especially after 20 minutes of exposure to LPS, exhibited very little phosphorylation of S21/9 but comparable phosphorylation of the activating Y279/216. (B) CTR-DCs displayed increased β-catenin and little GSK in the nucleus compared with RAPA-DCs. (C) Treatment of murine RAPA-DCs with the GSK-3 inhibitor LiCl (10mM) before LPS stimulation profoundly inhibited IL-12p70 secretion after TLR4 ligation in both CTR- and RAPA-DCs (mean ± SD). *P < .05. The data shown are representative of more than 3 experiments performed.