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. Author manuscript; available in PMC: 2021 Aug 20.
Published in final edited form as: Nat Struct Mol Biol. 2021 Mar 8;28(3):300–309. doi: 10.1038/s41594-021-00568-8

Figure 5 ∣. FANCD2 and FANCI are ubiquitinated sequentially.

Figure 5 ∣

a, Ubiquitination time course of ID (1 μM) by recombinant Core complex (0.1 μM) and UBE2T (1.5 μM). The positions of substrates and products are marked. b, Reaction time course comparing the ID complex, a complex assembled with ubiquitinated FANCD2 and non-ubiquitinated FANCI (IDnonUb:Ub), and the individual proteins (all at 1 μM). c, Reaction time course comparing the ID complex, a complex assembled with FANCI harboring the R1285Q FA mutation, individual FANCI, and the individual FANCI R1285Q mutant (all at 1 mM). a-c, The loading controls for substrates of the reactions are marked by arrows. Reactions were run on the same SDS-PAGE gel for direct comparison. They were visualized by Coomassie staining (top panels) or immunoblotting with a ubiquitin-specific antibody (bottom panels). The experiments were independently repeated three times with similar results. Uncropped blot and gel images are shown in the Supplementary Fig. 3.