Figure 8. FBXL14 mediates ubiquitination and degradation of c-Myc, whereas USP13 stabilizes c-Myc protein through deubiquitination. (A) Ubiquitination (Ub) assay showing that overexpression of FBXL14 promoted degradation of c-Myc in GSCs. GSCs (T387) were transduced with Flag-FBXL14 or vector control through lentiviral infection for 48 h, treated with the proteasome inhibitor MG132 for 6 h, harvested for IP of c-Myc with an anti–c-Myc antibody, and then immunoblotted with anti-ubiquitin antibody. Total cell lysates (TCL) were also immunoblotted with antibodies against FBXL14, USP13, c-Myc, and tubulin. (B) Ubiquitination assay showing that FBXL14 knockdown decreased c-Myc ubiquitination in GSCs. GSCs (T387) were transduced with shFBXL14 or shNT through lentiviral infection for 48 h and then treated with the proteasome inhibitor MG132 for 6 h before collecting samples. Cell lysates were immunoprecipitated with an anti–c-Myc antibody and then immunoblotted with anti-ubiquitin antibody. (C) Ubiquitination assay showing that FBXL14 mediated ubiquitination of wild-type c-Myc (lane 4) but not the ubiquitin-insensitive c-Myc mutants (Mt): T58A-Myc (lane 5), S62A-Myc (lane 6), and T58A-S62A-Myc (lane 7). Flag-FBXL14 or vector control and wild-type c-Myc or the c-Myc mutant were overexpressed in 293FT cells and then subjected to ubiquitination assay and IB analysis. (D) Ubiquitination assay showing that USP13 knockdown increased c-Myc ubiquitination and decreased c-Myc protein levels in GSCs. GSCs (T387) were transduced with shUSP13 (shUSP13-50 or shUSP13-52) or shNT control for 48 h and then treated with the proteasome inhibitor MG132 for 6 h before collecting samples. Cell lysates were immunoprecipitated with an anti–c-Myc antibody and then immunoblotted with anti-ubiquitin antibody. (E) Ubiquitination assay showing that c-Myc ubiquitination was specifically attenuated by wild-type USP13 but not catalytic dead mutant USP13 (C345A). GSCs (T387 or T4121) were transduced with Flag-USP13, Flag-USP13-C345A, or vector control for 48 h, treated with the proteasome inhibitor MG132 for 6 h, and then subjected to analysis of c-Myc ubiquitination and IB analyses. (F) Ubiquitination analysis showing that USP13-mediated deubiquitination antagonizes FBXL14-mediated c-Myc ubiquitination to regulate c-Myc protein levels in GSCs. GSCs (T387) were transduced with HA-FBXL14, Flag-USP13, Flag-USP13 plus HA-FBXL14, or vector control for 2 d through lentiviral infection, treated with MG132 for 6 h, and harvested for ubiquitination assay of c-Myc and IB analyses. FBXL14 overexpression alone increased c-Myc ubiquitination (lane 3), whereas USP13 overexpression alone decreased c-Myc ubiquitination (lane 4). However, USP13 and FBXL14 double overexpression attenuated the increased c-Myc ubiquitination induced by FBXL14 overexpression (lane 5). (G) Ubiquitination assay showing that c-Myc protein level was regulated by USP13-mediated deubiquitination and FBXL14-induced ubiquitination in GSCs. GSCs (T387) were differentiated for 2 d and transduced with shUSP13, shFBXL14, shUSP13 plus shFBXL14, or shNT for 2 d through lentiviral infection, treated with MG132 for 6 h, and then collected for ubiquitination analysis and IB analyses. USP13 knockdown alone increased c-Myc ubiquitination (lane 3), whereas FBXL14 knockdown alone decreased c-Myc ubiquitination (lane 4). However, USP13 and FBXL14 double knockdown attenuated the increased c-Myc ubiquitination induced by USP13 knockdown (lane 5). Mass is shown in kilodaltons.
Figure 8.

FBXL14 mediates ubiquitination and degradation of c-Myc, whereas USP13 stabilizes c-Myc protein through deubiquitination. (A) Ubiquitination (Ub) assay showing that overexpression of FBXL14 promoted degradation of c-Myc in GSCs. GSCs (T387) were transduced with Flag-FBXL14 or vector control through lentiviral infection for 48 h, treated with the proteasome inhibitor MG132 for 6 h, harvested for IP of c-Myc with an anti–c-Myc antibody, and then immunoblotted with anti-ubiquitin antibody. Total cell lysates (TCL) were also immunoblotted with antibodies against FBXL14, USP13, c-Myc, and tubulin. (B) Ubiquitination assay showing that FBXL14 knockdown decreased c-Myc ubiquitination in GSCs. GSCs (T387) were transduced with shFBXL14 or shNT through lentiviral infection for 48 h and then treated with the proteasome inhibitor MG132 for 6 h before collecting samples. Cell lysates were immunoprecipitated with an anti–c-Myc antibody and then immunoblotted with anti-ubiquitin antibody. (C) Ubiquitination assay showing that FBXL14 mediated ubiquitination of wild-type c-Myc (lane 4) but not the ubiquitin-insensitive c-Myc mutants (Mt): T58A-Myc (lane 5), S62A-Myc (lane 6), and T58A-S62A-Myc (lane 7). Flag-FBXL14 or vector control and wild-type c-Myc or the c-Myc mutant were overexpressed in 293FT cells and then subjected to ubiquitination assay and IB analysis. (D) Ubiquitination assay showing that USP13 knockdown increased c-Myc ubiquitination and decreased c-Myc protein levels in GSCs. GSCs (T387) were transduced with shUSP13 (shUSP13-50 or shUSP13-52) or shNT control for 48 h and then treated with the proteasome inhibitor MG132 for 6 h before collecting samples. Cell lysates were immunoprecipitated with an anti–c-Myc antibody and then immunoblotted with anti-ubiquitin antibody. (E) Ubiquitination assay showing that c-Myc ubiquitination was specifically attenuated by wild-type USP13 but not catalytic dead mutant USP13 (C345A). GSCs (T387 or T4121) were transduced with Flag-USP13, Flag-USP13-C345A, or vector control for 48 h, treated with the proteasome inhibitor MG132 for 6 h, and then subjected to analysis of c-Myc ubiquitination and IB analyses. (F) Ubiquitination analysis showing that USP13-mediated deubiquitination antagonizes FBXL14-mediated c-Myc ubiquitination to regulate c-Myc protein levels in GSCs. GSCs (T387) were transduced with HA-FBXL14, Flag-USP13, Flag-USP13 plus HA-FBXL14, or vector control for 2 d through lentiviral infection, treated with MG132 for 6 h, and harvested for ubiquitination assay of c-Myc and IB analyses. FBXL14 overexpression alone increased c-Myc ubiquitination (lane 3), whereas USP13 overexpression alone decreased c-Myc ubiquitination (lane 4). However, USP13 and FBXL14 double overexpression attenuated the increased c-Myc ubiquitination induced by FBXL14 overexpression (lane 5). (G) Ubiquitination assay showing that c-Myc protein level was regulated by USP13-mediated deubiquitination and FBXL14-induced ubiquitination in GSCs. GSCs (T387) were differentiated for 2 d and transduced with shUSP13, shFBXL14, shUSP13 plus shFBXL14, or shNT for 2 d through lentiviral infection, treated with MG132 for 6 h, and then collected for ubiquitination analysis and IB analyses. USP13 knockdown alone increased c-Myc ubiquitination (lane 3), whereas FBXL14 knockdown alone decreased c-Myc ubiquitination (lane 4). However, USP13 and FBXL14 double knockdown attenuated the increased c-Myc ubiquitination induced by USP13 knockdown (lane 5). Mass is shown in kilodaltons.

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