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Figure S3. The RING domain of SHPRH promotes PCNA polyubiquitination in RAD18-dependent manner. (A) RAD18 and RAD6 exclusively promote the monoubiquitination of PCNA. HEK 293T cells were transfected with 0.5 µg HA-Ub, and 0.5 µg FLAG-PCNA, along with 2.0 µg RAD18-myc-His, 50 ng UBC13-HA, 50 ng MMS2-HA, and 100 ng RAD6-HA, as indicated in the boxes. Anti-FLAG (PCNA) immunoprecipitates were analyzed with an anti-HA (Ub) antibody to visualize the ubiquitinated species of PCNA. (B) Schematic representation of the SHPRH (ΔRING) mutant (top). The E3 ligase activity of SHPRH for PCNA polyubiquitination is perturbed by a C-terminal deletion (ΔRING) (bottom). (C) SHPRH does not promote the polyubiquitination of c-JUN. To determine the substrate specificity of SHPRH, the His₆-c-JUN protein was expressed along with HA-Ub, SHPRH, UBC13, and MMS2, as indicated in the boxes. Unlike the enhancement of polyubiquitination in the His-₆-PCNA protein, the basal level of polyubiquitination of the His₆-c-JUN protein was not affected by the overexpression of SHPRH, UBC13, and MMS2.