Fig. 5

Depletion of PSMC3 inhibits USP14 upregulation of AGO2 proteins. a AGO2 degradation via proteasome pathway. After Hela cells were treated with the indicated doses of chloroquine for 18 h, AGO2 protein levels were measured by western blotting assays (left). HeLa cells were incubated with the proteasome inhibitor MG132 (30 µM) or with DMSO for 10 h. Whole-cell lysates were immunoprecipitated with anti-AGO2 antibody and analyzed with anti-ubiquitin, anti-GAPDH, and anti-AGO2 antibodies (right). b,c Depletion of PSMC3 facilitates AGO2 ubiquitination. b HeLa cells were transfected with the indicated plasmids. After 36 h, cells were treated with or without 30 µM MG132 for 10 h. AGO2 protein levels were analyzed by western blotting assays. c HeLa cells were transfected with control or PSMC3 siRNAs. After 36 h, cells were incubated with MG132 (30 µM) for 10 h. Whole-cell lysates were immunoprecipitated with anti-AGO2 antibody and analyzed with anti-ubiquitin, anti-GAPDH, and anti-AGO2 antibodies. d The PPI network analysis of PSMC3 by the STRING database. e The top 20 biological process (BP) terms in the enrichment analysis of the PSMC3-interacting proteins. f,g PSMC3 (f) and AGO2 (g) both interact with USP14. HeLa cells were co-transfected with the indicated plasmids. Immunoprecipitation assays were performed with anti-HA antibody and western blotting with anti-Flag antibody. h,i AGO2 is deubiquitinated by USP14. h HeLa cells were transfected with the indicated plasmids. After 36 h, cells were treated with (bottom) or without (top) 30 µM MG132 for 10 h. AGO2 protein levels were analyzed by western blotting assays. i HeLa cells were transfected with the indicated plasmids. After 36 h, cells were incubated with MG132 (30 µM) for 10 h. Whole-cell lysates were immunoprecipitated with anti-AGO2 antibody and analyzed with anti-ubiquitin, anti-GAPDH, and anti-AGO2 antibodies. j HeLa cells were treated with or without IU1 (an inhibitor of the enzymatic activity of USP14) and MG132 (30 µM) for 10 h. Whole-cell lysates were immunoprecipitated with anti-AGO2 antibody and analyzed with anti-ubiquitin, anti-GAPDH, and anti-AGO2 antibodies. k Depletion of PSMC3 inhibits USP14 upregulation of AGO2 proteins. HeLa cells were transfected with the indicated plasmids and then AGO2 protein levels were analyzed by western blotting assays after 48 h. l HeLa cells were transfected with either the pcDNA3 vector only or the indicated Flag-tagged PSMC3 (1–214) and siRNA-resistant PSMC3 (97–439) mutant. At 24 h after transfection, cells were split into two aliquots and were retransfected with control or PSMC3 siRNA. Cell lysates were analyzed by western blotting with anti-AGO2 and anti-GAPDH antibodies. m The interaction between AGO2 and USP14 is diminished in PSMC3-depletion cells. HeLa cells were cotransfected with the indicated plasmids. Immunoprecipitation assays were performed with anti-HA antibody and western blotting with anti-Flag antibody. All results are representative of three independent experiments