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. 2018 Nov 2;293(52):20099–20111. doi: 10.1074/jbc.RA118.005802

Figure 4.

Figure 4.

Characterization of lysine residues of FOXP3 targeted by RNF31. A, immunoblot analysis for the identification of lysine residues of FOXP3 targeted by RNF31 in the HEK-293T cell lysate transfected with FLAG-RNF31, His-ubiquitin, HA–tagged WT FOXP3, and mutant FOXP3 (with retention of one lysine residue). B, immunoblot analysis of ubiquitin chain formation on FOXP3 in HEK-293T cell lysate transfected with FLAG-RNF31, His-ubiquitin, HA–tagged WT FOXP3, and mutant FOXP3–8R/20R (with 8 lysine-to-arginine substitutions as indicated above, or with 20 lysine-to-arginine substitutions). C, immunoblot analysis for identifying RNF31 enzymatically inactive mutants in the HEK-293T cell lysate transfected with FLAG-FOXP3, Myc–tagged WT RNF31, or mutant RNF31 as indicated. D, immunoblot analysis of ubiquitin chain formation on FOXP3 in HEK-293T cell lysates transfected with FLAG-FOXP3, His-Ubiquitin, Myc–tagged WT RNF31 or N1 RNF31 truncation mutant as indicated. E, immunoblot analysis of ubiquitin chain formation on FOXP3 in a cell–free system with His6-E1 (100 ng), His6-E2 (200 ng), His6-FLAG ubiquitin (10 μg), StrepII FOXP3 (500 ng), 2× FLAG RNF31 (500 ng), 2× FLAG-N1-RNF31 (500 ng), and 2× FLAG RBCK1 (500 ng) being mixed in the reaction buffer, which was terminated by adding SDS protein loading buffer.