FIGURE 6.

ASXM enhances BAP1 binding to ubiquitin. A, recombinant His-BAP1 (1.6 μg, 20 nm) and MBP-ASXM2 (2 μg, 30 nm) were incubated with either GST or GST-ubiquitin-agarose beads (3 μg, 80 nm), and the pulldown fractions were analyzed by immunoblotting. B, recombinant His-BAP1 (1.6 μg, 20 nm) and GST-ASXM1 or GST-ASXM2 (2 μg, 40 nm) were incubated with ubiquitin-agarose beads, and the pulldown fractions were analyzed by immunoblotting. C, multiple sequence alignment between the ASXM domains of human ASXL1/2, Drosophila ASX, and other paralogs and orthologs of ASX. The mutants of ASXM2, including the cancer-associated mutants used in F–H and Fig. 9, are shown. D, GST pulldown interaction assay and in vitro DUB reactions of H2A using His-BAP1 and GST-ASXM2 (full-length and deletion mutant forms). For the pulldown assay, His-BAP1 (1.6 μg, 20 nm) was incubated with GST-ASXM2 (2 μg, 40 nm) or the different fragment of GST-ASXM2 (2 μg, 50 nm). His-BAP1 (8 ng, 2 pm) and the different recombinant ASXM2 fragments (10 ng, 4 pm) were used for the DUB reactions. E, His-BAP1 (1.6 μg, 20 nm) and the different GST-fused fragments of ASXM2 (2 μg, 40 nm) were subjected to ubiquitin-agarose pulldown assay followed by immunoblotting. F, MBP-pulldown interaction assay using recombinant MBP-ASXM2 (full-length and mutant forms) (2 μg, 30 nm) and His-BAP1 (1.6 μg, 20 nm). G, GST-ubiquitin pulldown assay using MBP-ASXM2 full-length and the different mutant forms with His-BAP1. The pulldown was done as in A. H, in vitro DUB reactions of H2A using His-BAP1 (8 ng, 2 pm) and the different recombinant MBP-ASXM2 (10 ng, 2.8 pm).