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. 2019 May 28;116(24):11754–11763. doi: 10.1073/pnas.1820990116

Fig. 1.

Fig. 1.

Identification of SCFFBXO22 as HDM2 E3 II. (A) HeLa cell proteome contains at least five peaks of HDM2 E3 ligase activity. Aliquots of the indicated Mono-Q fraction (1 µL) were assayed for E3 activity to support the ubiquitination of GST-HDM2 C464A, which is described in detail in SI Appendix, Methods. Lane 1 is the reaction with HeLa cell extract (10 µg). Note that flow-through to Mono-Q was found to contain little E3 activity. (B) Identification of FBXO22 by MS/MS analysis. MS identified 20 unique peptides that cover 44.4% of the FBXO22 sequence (Top). (Bottom) Representative MS/MS spectrum showing a tryptic FBXO22 peptide (m/z 867.46782+) with the sequence identified as HQLTEVGLLDNPELR. (C) SCFFBXO22 supports the ubiquitination of HDM2. HDM2 E3 II (Superose 6 fraction 27, 0.66 µg) or SCFFBXO22 (0.3 pmol; purity shown in SI Appendix, Fig. S5) was assayed for E3 activity to support the ubiquitination of GST-HDM2 C464A.