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. 2015 Oct 16;25(11):1205–1218. doi: 10.1038/cr.2015.121

Figure 1.

Figure 1

ASXL1 forms an H2A deubiquitylation complex by interacting with BAP1. (A) Summary of the peptides identified by mass spectrometry from a FLAG-HA tandem purification of FLAG-HA-ASXL1 expressed in Flp-In T-REX 293 cells. Mass (Da), observed number of peptides and the score obtained by Mascot database searching (Matrix Science, UK) were indicated for each protein. (B) Co-IP of endogenous ASXL1 and BAP1 proteins in myeloblastic leukemia cells M1. The input lanes contain 5% of the amount used for IP. The ASXL1 and BAP1 mouse monoclonal antibody, SUZ12 rabbit monoclonal antibody were used for IP. HA was used as negative controls in IP. (C) Summary of the peptides that are in common identified by MS from the purifications of FH-ASXL1, FH-BAP1 and FH-HCF1 expressing 293 cells. (D) ASXL1 stimulates H2A deubiquitylation activity of BAP1 in vitro. In vitro deubiquitylation assays were performed using the indicated amounts of FLAG-tagged BAP1 recombinant protein with or without FLAG-tagged ASXL1. Nucleosomes purified from HeLa cells were used as substrates. After incubation for 1 h, western blot analyses were carried out with the indicated antibodies. (E) ASXL1 stimulates H2A deubiquitylation activity in vivo. Levels of H2AK119ub1 and H3K27me3 in ASXL1-mutated NOMO1 cells with or without ectopic expression of ASXL1. Histone H3 was used as a loading control.