Fig. 2

The ASXL subunits link MBD5, MBD6 to BAP1 via their C-terminal PHD fingers. A IP of endogenous MBD5 or MBD6 from wild-type or BAP1 knockout HEK293T cells followed by IB for BAP1, ASXL1, and ASXL2. IgG was used as a negative control, n = 2. B IP of endogenous MBD5 or MBD6 from ASXL1/2/3 triple-knockout (TKO) HEK293T cells followed by IB for BAP1, ASXL1, ASXL2, MBD5, and MBD6. IgG was used as a negative control, n = 2. C Schematic diagram depicting the truncations of human ASXL1 protein. D HEK293T cells were transfected with plasmids expressing GFP, GFP-tagged full-length, or truncated ASXL1. The levels of GFP-tagged proteins were determined by western blot analysis, n = 2. E, F HEK293T cells were co-transfected with plasmids expressing each fragment of ASXL1 and either Halo-tagged MBD5 (E) or MBD6 (F). Then, the cells were subjected to co-IP assay using GFP-trap agarose followed by western blotting using antibodies against BAP1 and Halo-tag, n = 2. G Schematic diagram depicting the truncations of C-terminus of human ASXL1 protein. H HEK293T cells were transfected with plasmids expressing either GFP, GFP-tagged full-length, or GFP-tagged truncated C-terminus of ASXL1 and the protein levels of GFP-tagged proteins were determined by western blot analysis, n = 2. I, J HEK293T cells were co-transfected with plasmids expressing GFP-tagged truncated C-terminus of ASXL1 and either Halo-tagged MBD6 (I) or MBD5 (J). They were subjected to co-IP assay using an antibody against GFP followed by western blotting using antibodies against Halo-tag, n = 2. K CLUSTALW alignment shows the similarity between PHD fingers of ASXL1/2/3. L HEK293T cells were transfected with GFP-tagged PHD fingers of ASXL1/2/3 followed by IP of GFP and IB of MBD5, MBD6, and BAP1, n = 2. M The GFP-fusion proteins were purified from HEK293T cells transduced with GFP-tagged PHD finger of ASXL1/2/3. The purified proteins were subjected to mass spectrometry analysis. Peptide numbers of representative proteins were shown. N Schematic diagram depicting the PHD finger depletion of human ASXL1/2 proteins targeted by sgRNA. O, P IP of MBD6 from wild-type cells and ASXL1-PHD finger deleted (O) or ASXL2-PHD finger deleted (P) cells followed by IB of MBD6 and ASXL1 (O) or ASXL2 (P), n = 2