Figure 2. IL-1R-mediated GSK3-α phosphorylation is independent on PI3K-AKT kinase activity.

(A) Lysates from 293-IL-1R cells were untreated or treated for 15 or 30 min with IL-1 (1 ng/ml) were immunoprecipitated with anti-AKT, anti-GSK3α and β, followed by protein blot analysis. WCL (whole cell lysates). (B) GSK3α was pulled down with two rounds of immunoprecipitation. Immunoprecipitated complex, whole cell lysate and the remaining supernatant were analyzed by western blot analysis using GSK3α and Akt1 antibody. (C) Wild-type Th17 cells were untreated or treated with IL-1 (10ng/ml) for different time points in the presence and absence of 1μM Akt inhibitor (Akt IV). Cell lysates were analyzed by western blot analysis using antibodies as indicated. Densitometry quantitation of pooled data is plotted below. (D) Wild-type bone-marrow macrophages were untreated or treated with insulin (10nM) for different time points in the presence and absence of 1μM Akt inhibitor (Akt IV), cell lysates were analyzed by western blot analysis using antibodies as indicated. (E) Cell lysates from wild-type and Gsk3a−/− Th17 cells untreated or treated with IL-1 (10ng/ml) for different time points were analyzed by western blot analysis using antibodies as indicated. (F) Wild-type and Gsk3a−/− Th17 cells were rested for overnight, followed by incubation with 10ng/ml IL-1 or IL-2 for three additional days and with ³H for one more day for thymidine incorporation experiment. (G) Naïve wild-type, Gsk3a−/−CD4⁺ T cells (CD4⁺CD44^low) were polarized to Th17 cells in the presence and absence of IL-1β, followed by intracellular cytokine staining for IL-17 and IFN- γ (H) Real-time PCR analysis of relative expression of IL-17, IL-21, IL-23R, and ROR-γt in wild-type and Gsk3a−/− Th17 cells as compared to the Th0 cells. Error bars (F–H), s.d. **, p<0.01 (two tailed t-test). All data are representative of three independent experiments.