Fig. 3
Direct stimulation of EGFR induces the cellular cascade involved in GPCR desensitization. A, B, D EGF (+) represents the cells treated with 10 ng/mL EGF for 20 min; EGF (w/+) represents the cells treated with EGF for 5 min, washed, and rechallenged with EGF for 5 min. A HEK-293 cells transfected with HA-Src. Cell lysates were immunoblotted with antibodies against p-Src (Y416) and HA. *p < 0.05, **p < 0.01 compared with the “−” group (n = 4). B HEK-293 cells transfected with FLAG-Akt. Cell lysates were immunoprecipitated with FLAG beads, and IPs were immunoblotted with antibodies against p-Akt (T308) and FLAG, respectively. **p < 0.01, #p < 0.05 compared with the “−” and “ + ” group, respectively (n = 3). C HEK-293 cells transfected with HA-USP33 and FLAG-Akt were treated with 10 ng/mL EGF for 5 min or 20 min. Cell lysates were immunoprecipitated with FLAG beads. Co-IP/lysate and IPs were immunoblotted with antibodies against HA and FLAG. **p < 0.01 compared with the “0” min group (n = 4). D HEK-293 cells expressing D3R were transfected with HA-Ub and FLAG-arrestin3. Cells were treated with EGF, followed by the ubiquitination assay. The lower panel (IP) displays the immunoprecipitated total FLAG-arrestin3, while the upper panel shows the ubiquitin-conjugated arrestin3. **p < 0.01, #p < 0.05 compared with “1” and “3 or 4” groups, respectively (n = 3)