Extended Data Fig. 3: In vitro reconstitution of human MPU from HEK293F cells.
(a) Representative SEC (Superose 6 Increase 10/300) of the human MPU complex purified from 293F cells. More than five biologically independent experiments were performed (n > 5). The column was calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa). The black box highlights the referenced protein from the bands of the MPU member, representing the relative mobility of endogenous UBE2D paralogs in HEK293F cells, as identified by mass spectrometry. The sequences of the unique peptides identified in the proteomic analysis are highlighted in red. The detailed information was described in Supplementary Table 3. (b) Protein identity matrix of UBE2D from humans and mice generated via the UniProt alignment tool. (c) In vitro reconstitution of human PADI6-UHRF1-UBE2D2. Purified Strep-hUHRF1 (SRA-RING construct) was incubated separately with His-hPADI6 and His-hUBE2D2, as well as in a combined mixture of all three proteins. The mixtures were pulled down using Strep resin. SDS-PAGE analysis of the eluates and input samples was performed to separate proteins, which were stained with Coomassie blue to assess their interactions. Three biologically repeated experiments were performed. (d) Flowchart of cryo-EM data processing for the complex of second peak contains nucleosomes. Details are described in the Methods section.
