Fig. 3

MBD5 and MBD6 evolutionally contributes to the stability of the BAP1 complex. A Alignment by CLUSTALW analysis shows the similarity between MBD domains of human MBD5 and MBD6. B HEK293T cells were co-transfected with plasmids expressing GFP-tagged PHD finger of ASXL1 and each Halo-tagged truncated MBD domain of MBD6. Then the cells were subjected to co-IP assay using GFP-trap agarose followed by IB for Halo-tag, n = 2. C HEK293T cells were transfected with plasmids expressing Halo-tagged MBD6-WT, MBD6-Δ12aa, or MBD6-K61A/C66A together with GFP-tagged ASXL1 followed by IP of GFP and IB for Halo-tag, n = 2. D HEK293T cells were transduced with lentivirus expressing GFP, GFP-tagged MBD6-WT, MBD6-Δ12aa, or MBD6-K61A/C66A. The protein levels of ASXL1, ASXL2, and BAP1 were determined by western blot. HSP90 was used as an internal control, n = 2. E, F HEK293T cells stably expressing GFP, GFP-tagged MBD5, or GFP-tagged MBD6 were treated with CHX (50 μg/ml) for indicated time durations. The whole cell lysate was collected at each time point, and the total protein level of BAP1 was determined by western blot, n=2 (E) and quantified by ImageJ (F). G The computational BLAST with the 12-aa from human MBD5 and MBD6 in Drosophila protein database. H Schematic diagram depicting human MBD5 and MBD6 and isoforms of Drosophila SBA. I The GFP-fusion proteins were purified from Drosophila S2 cells transduced with the GFP-tagged MBD domain of SBA. The purified proteins were subjected to mass spectrometry analysis. Peptide numbers of representative proteins pulled down by the GFP-MBD domain of SBA were shown. JDrosophila S2 cells were subjected to co-IP assay using an antibody against Calypso followed by IB for ASX and SBA, n = 2. KDrosophila S2 cells were subjected to co-IP assay using an antibody against SBA followed by IB for SBA and ASX, n = 2. LDrosophila S2 cells were subjected to co-IP assay using an antibody against ASX followed by IB for ASX and SBA, n = 2. M The western blot shows protein levels of SBA, ASX, and Calypso in Drosophila S2 cells treated with dsRNA targeting SBA. Histone H3 was used as internal control, n = 2. N Schema of the Drosophila PR-DUB complex and the human BAP1 complex