Xenbase Image ID: 170840

Figure S2. MutSa and PCNA mutants cloned and purified, Related to Figure 2 (A) Schematic of X.l. MSH6. The domain structure of this protein and the locations of mutations used in the study are illustrated. (B) The sequence of the conserved FXE domain in MSH6 is illustrated for several species. The amino acid substitutions used in this study are indicated in red. (C) The sequence of the conserved PIP domain in MSH6 is illustrated for several species. The triple amino acid substitution used in this study is indicated in red. (D) Purified MutSaWT, MutSaFXE, or MutSaPIP complex stained with Coomassie blue. (E) The sequence of the conserved interdomain connector loop of PCNA is illustrated for several species. The double amino acid substitution used in this study is indicated in red. (F) Purified PCNAWT and PCNAL126A,I128A stained with Coomassie blue. (G) Co-immunoprecipitation of purified recombinant WT and mutant MSH2-MSH6 and PCNA using preimmune and purified IgG- or MSH2-specific antibodies. Immunoprecipitates were analyzed by Western blot with the indicated antibodies (left panel). The combination of MSH2-MSH6 and PCNA mutants analyzed are indicated in the table (right panel). (H) PCNA immunodepletion. Mock- and PCNA-depleted HSS was analyzed by Western blot with the indicated antibodies to demonstrate depletion of PCNA. (I) Quantification of ICL repair in Mock-, PCNA-depleted, or PCNA-depleted HSS supplemented with recombinant PCNAWT, or PCNAL126A,I128A at 3 hrs. Results represent mean ± SEM of n=4 independent experiments. Image published in: Kato N et al. (2017) Copyright © 2017. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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