Fig. 7
Ubiquitination of AKT is required for its translocation to the cell membrane and subsequent phosphorylation of Mdm2. A CTRL-shRNA and TRAF6-KD cells were transfected with D3R and GFP-AKT. The cells were treated with 100 nM Quin for 5 min or 2 min, washed, and re-challenged. The scale bar represents 10 μm. The 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 Veh/CTRL-shRNA group, ####p < 0.0001 compared to w+/ CTRL-shRNA group (n = 5). B HEK293 cells expressing D3R were transfected with GFP-Mdm2 and FLAG-WT-AKT or FLAG-K8/14R-AKT. Then the cells were treated with 100 nM Quin for 5 min, washed, and re-challenged. ****p < 0.0001 compared to Mock group, ####p < 0.0001 compared to “WT/w+” group (n = 3). C Cells expressing D3R were transfected with GFP-WT-AKT or GFP-K8/14R-AKT and HA-Mdm2. Cells were treated with 100 nM Quin for 2 min, washed, and re-challenged. Cells were labeled with rabbit monoclonal antibodies against HA (1:1000 dilution), followed by incubation with Alexa 594-conjugated anti-rabbit antibodies (1:500 dilution). Arrows in the “w/+” panel represent colocalization of AKT and Mdm2. The scale bar represents 10 μm. D HEK293 cells expressing D3R were pretreated with 200 nM Triciribine for 6 h, followed by treatment with 100 nM Quin for 5 min, washed, and re-challenged with Quin for 5 min. Cell lysates were immunoblotted with antibodies against phospho-Mdm2 (S166) and Mdm2, respectively. ****p < 0.0001 compared to “Veh/−” group, ####p < 0.0001 compared to “Veh/w+” group (n = 3). E CTRL-shRNA and TRAF6-KD cells were transfected with D3R. Cells were treated with 100 nM Quin for 5 min washed, and re-challenged. Then cell lysates were immunoblotted with antibodies against phospho-Mdm2 (S166) and Mdm2, respectively. ****p < 0.0001 compared to “CTRL-shRNA/−” group, ####p < 0.0001 compared to “CTRL-shRNA/w+” group (n = 3). F CTRL-shRNA and TRAF6-KD cells were co-transfected with D3R and GFP-Mdm2. Cells were treated with 100 nM Quin for 2 min, washed, and re-challenged. Co-localization between Mdm2 and DAPI is shown as Pearson’s coefficient. ****p < 0.0001 compared to corresponding “CTRL-shRNA/Veh” group, ####p < 0.0001 compared to “CTRL-shRNA/w+” group (n = 11). The scale bar represents 10 μm