Based on previously described ubiquitination assays8,28, we set up a large-scale in vitro reconstitution of the FA core complex−mediated ubiquitination of D2–I. In a t… See more

Fluorescently labeled DNA (6FAM-labeled or Alexa647-labeled on the 3ʹ end, purchased from … See more

A volume of 300 µl of purified D2–I, FANCD2 and FANCI at 1 µM were sequentially run on a Superdex 200 10/300 column (GE Healthcare Life Sciences) equilibrate… See more

We used purified StrepII-tagged FANCD2 and His-tagged FANCI to probe their interaction. In a total volume of 50 µl per reaction, FANCD2:FANCI at molar ratios 1:0, 1:0.5, 1… See more

Three microliters of ~1 µM ubD2–I, D2–I or D2 complex was blotted on plasma-cleaned UltraAufoil R1.2/1.3 grids45 (Quantifoil) for 3–4.5 s and plunged into liquid ethane using a Vitr… See more