Fig. 5
TRAF6 enters the nucleus in an importin β1-dependent manner, which promotes AKT ubiquitination. (A-C) After transfected cells were treated with corresponding drugs, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. After being washed 3 times with PBS, cells were visualized by laser-scanning confocal microscope (TCS SP5/AOBS/Tandem; Germany). The scale bar represents 10 μm. “+” and “w/+” refer to drug-treated and washed/retreated cells, respectively. A HEK293 cells expressing D3R were transfected with GFP-TRAF6. The colocalization of TRAF6 with DAPI was determined by Pearson correlation coefficient. ****p < 0.0001 compared to vehicle-treated group (n = 8). B HEK293 cells were transfected with GFP-TRAF6 and receptors. The colocalization of TRAF6 with DAPI was determined by Pearson correlation coefficient. ***p < 0.001, ****p < 0.0001 compared to corresponding veh group (n = 6). C CTRL-shRNA and importin β1-KD cells were co-transfected with D3R and GFP-TRAF6. The cells were pretreated with either vehicle or 30 ng/ml LMB for 30 min, followed by treatment with 100 nM Quin for 5 min, washed, and re-challenged. The colocalization of TRAF6 with DAPI is shown as Pearson’s coefficient. ****p < 0.0001 compared to “CTRL-shRNA/veh” group, ####p < 0.0001 compared to “CTRL-shRNA/w+” group (n = 9). D CTRL-shRNA and importin β1-KD cells were co-transfected with D3R, HA-Ub and FLAG-AKT. Cell lysates were immunoprecipitated with anti-FLAG beads. Co-IP was immunoblotted with antibodies against HA. IP was immunoblotted with antibodies against FLAG. ****p < 0.0001 compared to “CTRL-shRNA/−” group (n = 3). E CTRL-shRNA and importin β1-KD cells were transfected with D3R. The cellular levels of cAMP were evaluated by using the CRE-luciferase reporter gene assay. CTRL-shRNA/Quin groups were significantly different from other groups at treatment concentrations of quinpirole between 3 × 10−10 and 10−8 M. (*p < 0.05, ***p < 0.001, n = 3)