Figure 5.

FOSL1 promotes CYLD SUMOylation to impair deubiquitination of NF-κB signaling intermediaries

(A–D) K63-linked polyubiquitin chains of TRAF2 (A), TRAF6 (B), RIP1 (C), and NEMO (D) were analyzed in PN 35 and PN 182 GSCs with indicated modifications. (E) IB analysis of CYLD protein expression in PN 35 and PN 182 GSCs expressing exogenous FOSL1 or vector control, with or without FOSL1 depletion. Arrow indicates additional slow-migrating band in FOSL1-transduced PN GSCs. α-Tubulin was used as internal control. (F) PN 35 and PN 182 GSCs were co-transfected with HA-CYLD and one of the Myc-tagged SUMO isoforms (SUMO1–4). Immunoprecipitated HA-CYLD was either incubated with anti-HA antibody (middle panel) or was probed for SUMOylation using anti-Myc antibody (top panel). The expression level of SUMO1–4 in the cell lysates is also shown (bottom panel). (G) PN 35 and PN 182 GSCs were transduced with FLAG-tagged FOSL1 or vector control. CYLD was immunoprecipitated and then incubated with anti-SUMO1 antibody or anti-CYLD antibody, respectively. (H) MES 21 and MES 505 GSCs were transduced with shCtrl or shFOSL1 (targeting the 3′ UTR), reconstituted with WT FOSL1 or vector control. CYLD was immunoprecipitated and then incubated with anti-SUMO1 antibody or anti-CYLD antibody, respectively. (I) HEK293T cells were transduced with FLAG-tagged WT CYLD or K40R CYLD (an unSUMOylatable mutant), together with HA-tagged FOSL1, Myc-tagged SUMO1, and HA-tagged UBC9. FLAG-tagged CYLD was immunoprecipitated and then incubated with anti-Myc antibody or anti-FLAG antibody, respectively. (J) PN 35 and PN 182 GSCs were transduced with FLAG-tagged FOSL1 or vector control, with or without UBC9 knockdown. CYLD was immunoprecipitated and then incubated with anti-SUMO1 antibody or anti-CYLD antibody, respectively.