Figure 6. TRAF6-mediated GSK3β ubiquitination at lysine 183 is critical for TLR3-dependent cytokine production.

(a) BMDMs were stimulated with 10 μg ml⁻¹ poly I:C for 10 min and subjected to immunoprecipitation with an anti-Ub antibody followed by western blotting with an anti-GSK3β antibody. (b) HEK293T cells transfected with HA-GSK3β and HA-Ub along with Flag-TRAF6 plasmids were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-HA antibody. (c) HEK293T cells were transfected with HA-GSK3β and HA-Ub along with TRAF6 (WT) or TRAF6 (C70A) plasmids. These experiments were performed as described in b. (d) Traf6^+/+ and Traf6^−/− 3T3 cells stimulated with 10 μg ml⁻¹ poly I:C for 10 min were subjected to immunoprecipitation with an anti-GSK3β antibody followed by western blotting with an anti-Ub antibody. (e) GSK3β proteins were incubated with E1, E2 and biotinylated-Ub (Bt-Ub) in the presence or absence of Flag-TRAF6 proteins for in vitro ubiquitination of GSK3β. Ubiquitination of GSK3β was analysed by western blotting with streptavidin-HRP. (f) HEK293T cells transfected with Ub and Flag-TRAF6 along with HA-GSK3β WT or various HA-GSK3β mutants were subjected to immunoprecipitation with an anti-HA antibody followed by western blotting with an anti-Ub antibody. (g) HEK293-TLR3 cells were transiently transfected with GSK3β (WT) or GSK3β (K183R) plasmids. The levels of IL-6, TNF-α and c-Fos mRNA were determined by real-time PCR analysis (top). GSK3β expression levels were confirmed by western blotting with an anti-HA antibody (bottom). A longer exposure of the HA blot shows the presence of ubiquitin ladder. Data are presented as the mean±s.d. from at least three independent experiments. Statistical analyses were calculated using the Student’s t-test (**P<0.01).