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. Author manuscript; available in PMC: 2018 Feb 16.
Published in final edited form as: Mol Cell. 2017 Feb 2;65(4):699–714.e6. doi: 10.1016/j.molcel.2017.01.008

Figure 6. Acidity at the N-terminus of E2 negatively regulates FANCL-mediated UBE2T ubiquitination of FANCD2.

Figure 6

(A) Structure of hUBE2T (slate) in complex with the FANCL RING domain (rose) (PDB: 4CCG), with selected residues shown as sticks.

(B) E1-E2 thioester transfer assays of the indicated proteins.

(C) FANCL-mediated UBE2T monoubiquitination of FANCD2 performed under multiple turnover conditions (top). UBE2T loading control (bottom).

(D) FANCL-mediated UBE2T monoubiquitination of FANCD2 performed under single turnover conditions (left). E2~Ub charging control (right).

(E) Multiple turnover FANCD2 monoubiquitination assay as in C (top). E2s were treated or untreated with lambda phosphatase, as indicated. Anti-pSer5 UBE2T western blot (bottom).

(F) Single turnover FANCD2 monoubiquitination assay as in D (top). Anti-pSer5 UBE2T western blot (bottom).