Extended Data Fig. 2: Recombinant TRAIP supports CMG unloading at cisplatin-ICLs.

a, NPE immunodepleted of TRAIP was loaded alongside a dilution series of mock-depleted NPE and analysed for TRAIP. A relative loading amount of 100 corresponds to 2 µl of NPE. Non-specifically detected proteins are marked with asterisks. Four independent experiments. b, Bacterially expressed wild-type rTRAIP, rTRAIP(R18C), His₆-SUMO-rTRAIP(wild type), and His₆-SUMO-rTRAIP(ΔPIP) (comprising residues 1–455) were partially purified, resolved by SDS–PAGE and visualized with Coomassie blue staining. Note that His₆-SUMO-rTRAIP is obscured by co-migrating, contaminating proteins. Bacterially expressed rTRAIP was used for all subsequent experiments, unless otherwise indicated. Results are typical of at least two independent purifications. c, Wild-type rTRAIP and rTRAIP(R18C) were expressed in Sf9 insect cells and purified using an N-terminal 3×Flag tag. The tag was then cleaved using 3C protease. The recombinant proteins, along with a buffer sample containing 3C protease only, were resolved by SDS–PAGE and visualized with Coomassie blue staining. Results are typical of at least three independent purifications. d, Mock-depleted and TRAIP-depleted extracts supplemented with wild-type rTRAIP or rTRAIP(R18C) used in Fig. 1b were analysed by immunoblotting for TRAIP. The absence of the non-specific bands seen in a may be due to shorter incubation with the TRAIP antibody. The concentration of added recombinant TRAIP relative to endogenous TRAIP fluctuates among experiments (for example, compare d and Extended Data Fig. 3b). We ascribe this difference to variations in non-specific removal of endogenous TRAIP from extracts during the mock-depletion procedure, and possibly also in the delivery of recombinant TRAIP into the extract. Seven independent experiments. e, pICL^Pt was replicated in mock-depleted or TRAIP-depleted extracts supplemented with [α-³²P]dATP and Sf9-expressed wild-type rTRAIP, rTRAIP(R18C) or 3C protease alone and analysed as in Fig. 1b. Two independent experiments. f, Extracts used in the replication reaction shown in e were analysed as in d. Two independent experiments. g, pICL^Pt was replicated in the indicated egg extracts with [α-³²P]dATP and analysed as in Fig. 1b. Two independent experiments. h, Extracts used in the replication reaction shown in g were analysed as in d. Note that deleting the C-terminal PIP box disrupts the epitope for the TRAIP antibody used for immunoblotting. Therefore, to assess the activity of TRAIP(ΔPIP) in ICL repair relative to wild-type TRAIP, His₆-SUMO-tagged proteins were added back to TRAIP-depleted extract and assayed in g. The relative amounts of His₆-SUMO-TRAIP(wild type) and His₆-SUMO-TRAIP(ΔPIP) were compared by detecting the histidine tag. By blotting the same extracts for TRAIP, a comparison of the relative concentrations of His₆-SUMO-TRAIP(ΔPIP) and endogenous TRAIP was made. Two independent experiments. i, Mock-depleted and TRAIP-depleted extracts used in the replication reactions shown in Fig. 1c, d were analysed as in d. Three independent experiments.