Fig. 8

Roles of AKT ubiquitination in β-arrestin2 deubiquitination under desensitization conditions. A Cells expressing D₃R were transfected with GFP-AKT. Cells were pretreated with 10 μM PP2 for 30 min, then treated with 100 nM Quin for 5 min, washed, and re-challenged. The scale bar represents 10 μm. Membrane localization of AKT was determined by Pearson correlation coefficient. Data show one representative example out of three independent experiments. ****p < 0.0001 compared to CTRL-shRNA/Veh group, ^####p < 0.0001 compared to CTRL-shRNA/w + group (n = 4). B HEK293 cells expressing D₃R were transfected with FLAG-Mdm2 and HA-AKT. Cells were pretreated with 10 μM PP2 for 30 min, then treated with 100 nM Quin for 5 min, washed, and re-challenged with Quin for 5 min. **p < 0.01 compared to Veh/veh group, ^###p < 0.001 compared to Veh/w + group (n = 3). C CTRL-shRNA and TRAF6-KD cells were transfected with D₃R, HA-Ub, and FLAG-β-Arr2. Then the cells were treated with 100 nM Quin for 5 min, washed, and re-challenged. ***p < 0.001 compared to “CTRL-shRNA/−” group (n = 3). D CTRL-shRNA and TRAF6-KD cells were transfected with D₃R, GFP-β-Arr2, and FLAG-Gβ1. Then the cells were treated with 100 nM Quin for 5 min, washed, and re-challenged with Quin for 5 min. ****p < 0.0001 compared to “CTRL-shRNA/−” group (n = 3). E HEK293 cells expressing D₃R were transfected with HA-Ub and FLAG-β-Arr2. 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 Quin for 5 min. ****p < 0.0001 compared to “Veh/−” group (n = 3)