Fig. 6
Functional roles of Src in the nuclear import of TRAF6. A HEK293 cells expressing D3R were transfected with GFP-TRAF6. The cells were treated with 30 ng/ml LMB for 30 min, followed by treatment with either vehicle or 10 μM PP2 for 30 min. Thereafter, the cells were treated with 100 nM Quin for 5 min, washed, and re-challenged. Colocalization between TRAF6 and DAPI is shown as Pearson’s coefficient. ****p < 0.0001 compared to “Veh/veh” group (n = 8). The scale bar represents 10 μm. B HEK293 cells expressing D3R were transfected with HA-Ub and FLAG-AKT. The cells were pretreated with either vehicle or 10 μM PP2 for 30 min, followed by treatment with 100 nM Quin for 5 min, washed, and re-challenged with 100 nM Quin for 5 min. Co-IP and IP were immunoblotted with antibodies against HA and FLAG, respectively. ****p < 0.0001 compared to corresponding Veh/veh group, ####p < 0.0001 compared to Veh/ w + group (n = 3). C HEK293 cells expressing D3R were transfected with GFP-AKT and FLAG-TRAF6. The cells were pretreated with either vehicle or 10 μM PP2 for 30 min, followed by treatment with Quin, washed, and re-challenged with Quin. Co-IP/Lysate and IP were immunoblotted with antibodies against GFP and FLAG, respectively. ****p < 0.0001 compared to Veh/veh group, ####p < 0.0001 compared to Veh/ w + group (n = 3). D HEK293 cells expressing D3R were transfected with GFP-importin β1 and FLAG-TRAF6. The cells were pretreated with either vehicle or 10 μM PP2 for 30 min, followed by treatment with Quin, washed, and re-challenged with Quin. Co-IP/Lysate and IP were immunoblotted with antibodies against GFP and FLAG, respectively. ***p < 0.001 compared to Veh/veh group, ###p < 0.001 compared to Veh/w + group (n = 3). E HEK293 cells expressing D3R were pretreated with vehicle and 10 μM PP2 for 30 min. The cellular levels of cAMP were evaluated by using the CRE-luciferase reporter gene assay. Veh/Quin group was significantly different from other groups at treatment concentrations of Quinpirole between 10−9 and 10−8 M. (**p < 0.01, ***p < 0.001, n = 3)