Figure 3. Verification of RNF114 substrates.

1. RNF114 substrate validation. HEK293 cells were co‐transfected with constructs expressing FLAG‐tagged substrates, wild‐type or mutated His‐RNF114, and EGFP, and Western blot was performed with anti‐FLAG and anti‐His antibodies. The results show that the protein levels of CD74, TAB1, OPTN, TNIP1, APPL1, PSAT1, and RALGPS1 were reduced in the presence of wild‐type RNF114 compared to co‐transfecting with mutant RNF114.
2. RNF114‐dependent in vitro ubiquitination of substrates. Substrate ubiquitination assays were performed with purified RNF114, the reaction products were immunoblotted using anti‐His antibody to detect the ubiquitin (upper panel) or anti‐FLAG antibody detecting the substrate protein (middle panel), and the lower panel shows the merged signals. The results show that clear polyubiquitin chains were formed in the systems containing recombinant CD74, TAB1, TNIP1, PSAT1, or RALGPS1.

Data information: The relative intensity of Western blots was quantified and normalized, and the data are plotted at the bottom of the corresponding lanes.