Fig. 4.

Impaired UBE2J2 function enhances HLA-I downregulation by US2 via upregulation of TRC8. (A) US2-expressing TRC8-knockout cells were transduced with TRC8–Flag–ST2 or a control vector. To these cells, UBE2J2-targeting CRISPR gRNAs or empty vector were added. TRC8 levels were assessed by direct immunoprecipitation (IP). Upon UBE2J2 knockout, the expression of TRC8 increases. (B) US2-expressing TRC8-knockout cells were transduced with TRC8–Flag–ST2 or a control vector. To these cells, cDNA of wild-type (wt) or catalytically inactive (ci) HA-tagged UBE2J2 or a control vector were added. TRC8 levels were assessed by direct immunoprecipitation. Upon expression of catalytically inactive UBE2J2, the expression of TRC8 increases compared to that seen in control cells without HA–UBE2J2 or in cells with wild-type HA–UBE2J2. (C) Catalytically inactive UBE2J2 increases TRC8 levels in the US2 ERAD complex. A control vector, a vector expressing wild-type UBE2J2 (wt) or catalytically inactive UBE2J2 (ci) was transduced in U937 cells expressing eGFP–HLA-A2 and ST2–HA–US2. After G418 selection, cells were lysed in 1.0% digitonin lysis buffer, after which ST2–HA–US2 was immunoprecipitated by using StrepTactin beads. Immunoprecipitated complexes were eluted using d-Desthiobiotin, after which immunoblot analysis was performed for the proteins indicated. A short and a long exposure of the anti-HA immunoblot are shown.