Figure 3.

RNF31 mediates atypical ubiquitin conjugation of FOXP3. A, Myc-RNF31 and FLAG-FOXP3 were transfected into HEK-293T cells, 48 h later, cells were harvested and lysed for co-immunoprecipitation (co-IP) as indicated. B, human CD4⁺CD25^highCD127^low expanding Treg cells were cultured with anti-CD3/CD28 Ab in the presence of IL-2. Cells were collected for endogenous co-IP and Western blotting as indicated. C, in vitro expanded human Treg cells were incubated with specific antibodies. Representative confocal images were visualized for endogenous RNF31 (red) and FOXP3 (green) with DAPI used to visualize the nuclei (blue). D, FLAG-NEMO, FLAG-FOXP3, ubiquitin, and Myc-RNF31 were transfected into HEK-293T cells, followed by immunoblot analysis of linear ubiquitinated FLAG-FOXP3 or FLAG-NEMO as indicated. E, FLAG-FOXP3, Myc-RNF31, and His-ubiquitin were transfected into HEK-293T cells, cell lysates were incubated with nickel-nitrilotriacetic acid beads, followed by immunoblot analysis of ubiquitinated FLAG-FOXP3. F, FLAG-FOXP3, Myc-RNF31, and His-ubiquitin (WT) and mutant His-ubiquitin (KO, without retention of any lysine) were transfected into HEK-293T cells, cell lysates were incubated with nickel-nitrilotriacetic acid beads, followed by immunoblot analysis of ubiquitinated FLAG-FOXP3.