Fig 1: UBE2O is an E3 ubiquitin that induces polyubiquitination of ASXL1-MT. a Schematic experimental procedures of nano-LC-MS/MS analysis. b Venn diagram shows the overlap of proteins bound to ASXL1-MT in the conditions with or without coexpression of BAP1. Eighty-eight proteins, including UBE2O, bound to ASXL1-MT only with the coexpression of BAP1. See also Supplementary Data 3 and 4. c 293T cells were transfected FLAG-ASXL1-MT, MYC-UBE2O (WT), and MYC-UBE2O-C885S (CS). Total cell lysates were immunoprecipitated with anti-FLAG M2 antibody, and the ubiquitinated ASXL1-MT was detected with anti-ubiquitin antibody. d 293T cells were transduced with Cas9 and a vector control or three independent sgRNAs targeting UBE2O. Cells were then transfected with FLAG-ASXL1-MT and HA-BAP1. Cell lysates were subjected to western blotting analysis. The band intensities of FLAG relative to alpha-tubulin are shown. The value of FLAG/alpha-tubulin for ASXL1-MT alone without UBE2O depletion was set to 1. e 293T cells were transfected with FLAG-ASXL1-MT, HA-BAP1 (WT), HA-BAP1-C91S (CS), and Myc-ubiquitin. Total cell lysates were immunoprecipitated with anti-FLAG M2 antibody, and the ubiquitinated ASXL1-MT was detected with anti-Myc. Experiments shown in (c–e) were independently repeated at least three times with similar results. Shown are representative results