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Figure EV4. XPF‐ERCC1 mutant complexes are active in NER Mock‐depleted and XPF‐ERCC1‐depleted (ΔXE) high‐speed supernatant (HSS) egg extracts used in Fig 3B were analyzed by Western blot using α‐XPF antibodies. A dilution series of undepleted NPE was loaded on the same blot to determine the degree of depletion. A relative volume of 100 corresponds to 0.2 μl of NPE. *, background band. Mock‐depleted, XPF‐ERCC1‐depleted (ΔXE), and XPF‐ERCC1‐depleted HSS complemented with wild‐type (XEWT) or mutant XPF‐ERCC1 (XEMUT) were analyzed by Western blot using α‐XPF antibodies. *, background band. Untreated or UV‐treated (10 J/m2, left panel, 350 J/m2 right panel) plasmid DNA was incubated in HSS for 2 h. Samples were taken at time 0 and 2 h, and DNA was extracted and analyzed by an enzyme‐linked immunosorbent assay (ELISA) for the presence of CPDs. The highest value within one experiment was set to 100%. Error bars represent s.e.m. of three independent experiments. **P = 0.0061, paired t‐test. ns, not significant. Mock‐depleted, PCNA‐depleted (ΔPCNA), and PCNA‐depleted HSS complemented with recombinant His‐xlPCNA (ΔPCNA + PCNA) were analyzed by Western blot using α‐PCNA antibodies (Kochaniak et al, 2009). A dilution series of undepleted NPE was loaded on the same blot to determine the degree of depletion. A relative volume of 100 corresponds to 0.2 μl of NPE (top panel). These extracts were incubated with untreated or UV‐treated (350 J/m2) plasmids for 0 or 2 h at room temperature in the presence of 32P‐α‐dCTP. Reaction products were isolated, linearized with HincII, and separated on a 0.8% agarose gel. The DNA was visualized by autoradiography to show incorporation of 32P‐α‐dCTP during UDS (bottom left panel). The signal was quantified, the background signal from non‐damaged plasmid was subtracted, and the signal for the mock depletion condition was set to 100% to normalize the data. Error bars represent s.e.m. of three independent experiments. **P = 0.0059, paired t‐test compared to the mock condition. ns, not significant. As in (D) but using mock‐depleted or XPA‐depleted HSS. **P = 0.0014. Source data are available online for this figure. Image published in: Klein Douwel D et al. (2017) © 2017 The Authors. This image is reproduced with permission of the journal 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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