Fig. 3

GECX-MS to capture and identify UBE2D3 interacting with PCNA. a Structure of the substrate protein yPCNA (PDB ID: 4YHR) with its ubiquitination site K164 and two neighboring residues T163 and E165 shown in stick; structure of UbcH5C (PDB ID: 1 × 23), a yeast homolog of UBE2D3 (a human ubiquitin E2 enzyme), is shown at right with the active site residue Cys85 highlighted in stick. b Western blot analysis of in vitro crosslinking of UBE2D3 to yPCNA via the genetically encoded Uaa BprY or EB3 incorporated into yPCNA. An anti-His6 antibody was used to detect the His6 tag appended at the C-termini of yPCNA and UBE2D3. c Western blot of cell lysate of cells co-expressing yPCNA(165EB3) and UBE2D3, showing the in vivo crosslinking of UBE2D3 onto yPCNA by EB3. d SDS-PAGE gel of His-tag purified proteins from cells co-expressing yPCNA(165EB3) and UBE2D3. e Mass spectrum of cross-linked peptide between yPCNA and UBE2D3. U represents EB3 in the peptide sequence. f Mass spectrum of biotinylated cross-linked peptide between yPCNA and UBE2D3. U represents EB3 in the peptide sequence. g Extracted ion chromatography of biotinylated cross-linked peptide from input sample of biotin enrichment. RT: retention time; MA peak area, MH peak height. h Extracted ion chromatography of biotinylated cross-linked peptide from elute sample of biotin enrichment