Fig. 3

Determination of the subcellular localization of AKT and TRAF6. (A-D) 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 via a 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 were transfected with GFP-AKT or GFP-TRAF6. The colocalization of AKT or TRAF6 with DAPI was determined by Pearson correlation coefficient. ****p < 0.0001 compared to AKT transfection group (n = 8). B HEK293 cells were transfected with GFP-TRAF6. After 24 ~ 36 h, the cells were treated with 30 ng/ml LMB for 3 h. The colocalization of TRAF6 with DAPI was determined by Pearson correlation coefficient. *p < 0.05 compared to vehicle-treated group (n = 4). C HEK293 cells expressing D₃R were transfected with GFP-AKT. After 24 ~ 36 h, Cells were treated with 30 nM DA or 100 nM Quin for 5 min, washed, and re-challenged with 30 nM DA or 100 nM Quin for 5 min. ****p < 0.0001 compared to vehicle-treated group (n = 10). The membrane localization of AKT was determined by Pearson correlation coefficient. D HEK293 cells expressing D₃R were transfected with GFP-AKT. After 24 ~ 36 h, the cells were pretreated with 30 ng/ml LMB for 3 h, then treated with 100 nM Quin for 5 min, washed, and re-challenged with 100 nM Quin for 5 min. ****p < 0.0001 compared to Veh/veh treated group, ^####p < 0.0001 compared to Veh/w + group (n = 5). Membrane localization of AKT was determined by Pearson correlation coefficient. E HEK293 cells expressing D₃R were transfected with HA-Ub and FLAG-AKT. The cells were pretreated with either vehicle or 30 ng/ml LMB for 3 h, followed by treatment with 100 nM Quin for 5 min, washed, and re-challenged. ****p < 0.0001 compared to the corresponding Veh group (n = 3)