Fig. 2

ASXL3 directly interacts with BRD4 in small cell lung cancer cells. a The GFP-fusion proteins were purified from HEK293T cells transfected with GFP-tagged ASXL1/2/3. The purified proteins were subjected to mass spectrometry analysis. Peptide numbers of BAP1 complex subunits and BRD4 pulled down by GFP-ASXLs were shown. b BAP1, HCF1, FOXK1, and BRD4 levels in HEK293T cells 24 h after transfection with plasmids expressing GFP or GFP-ASXL1–3. Immunoprecipitation (IP) from whole cell lysates were performed with antibodies against the GFP epitope, followed by immunoblotting (IB) with antibodies against the indicated proteins. HSP90 was used as negative control, n = 3. c IP of endogenous ASXL3 of NCI-H1963 cells with two different homemade antibodies followed by IB for BAP1 and BRD4; IgG was used as negative control, n = 3. d IP of endogenous BRD4 from NCI-H1963 cells followed by IB for BRD4, ASXL3, and BAP1; IgG was used as negative control, n = 3. LC, light chain. e Nuclear extract from NCI-H1963 SCLC cells was subjected to size exclusion chromatography, and then protein levels of ASXL3, BAP1, and BRD4 were determined by western blot analysis. BRD2 and BRD3 were used as controls, n = 2. f Schematic diagram depicting the BBM depletion of human ASXL3 protein. g BAP1 and BRD4 levels in HEK293T cells 24 h after transfection with plasmids expressing GFP, GFP-ASXL3-FL, or GFP-ASXL3-ΔBBM. IP from whole cell lysates was performed with antibodies against the GFP epitope, followed by IB with antibodies against the BAP1 and BRD4, n = 3. h 293T cells were transfected with Flag-tagged BRD4 truncations (Additional file 2: Figure S3B) together with GFP-tagged ASXL3. Whole-cell lysates were used for IP with Flag antibody followed by IB for GFP. CDK9 was used as positive control, n = 3. i Recombinant His-tagged BRD4-ET domain and GST-tagged ASXL3-BBM was purified from Escherichia coli. The in vitro binding assay was performed to determine the direct interaction between BRD4-ET domain and ASXL3-BBM, n = 3. j Alignment by CLUSTALW analysis shows the similarity between ASXL3-BBM and BRD4 binding motif within other BRD4 binding proteins