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. Author manuscript; available in PMC: 2019 Apr 10.
Published in final edited form as: Nature. 2017 Nov 8;551(7680):389–393. doi: 10.1038/nature24484

Extended Data Figure 9. Characterization of the E3 ubiquitin ligase activity of RNF113A and TTDN1.

Extended Data Figure 9.

(a) TAP-RNF113A and the I264A RING finger mutant were stably expressed in HeLa-S cells and purified using anti-Flag resin. The eluted proteins were then analyzed by silver staining after SDS-PAGE (n=3 independent experiments). (b) Ubiquitin ligase assays using E1, E2 (UbcH5c plus Ubc13/MMS2; 50 nM each), and wildtype or I264A RNF113A. Reactions were analyzed by Western blot (n=3 independent experiments). (c) MMS sensitivity of lymphoblasts from two X-TTD patients in comparison to an unaffected individual (n=5 biological replicates; mean ± S.D.). (d) U2OS cells expressing the indicated combination of shRNA and RNF113A rescue vector were assessed for MMS sensitivity using MTS assay (n=5 technical replicates; mean ± S.D.). (e) Whole cell lysates of control or X-TTD lymphoblasts expressing indicated vectors after selection (n=2 independent experiments). (f) Immunofluorescence analysis of U2OS cells expressing the indicated shRNAs after MMS treatment. Western blot (n=2 independent experiments) from the same cells is shown on the bottom, as is the quantification of ASCC3 foci (n=3 biological replicates; mean ± S.D.).