Journal: Endocrinology

Article Title: AGEs-RAGE system down-regulates Sirt1 through the ubiquitin-proteasome pathway to promote FN and TGF-β1 expression in male rat glomerular mesangial cells.

doi: 10.1210/en.2014-1381

Figure Lengend Snippet: Figure 4. USP22 deubiquitinated Sirt1. A, The effects of AGEs (100 g/mL; 0, 3, 6, 12, and 24 hours) on USP22 protein expression. **, P .01; ***, P .001 vs 0 hour. B, GMCs were transfected with negative control, and 3 pairs of siRNA oligonucleotides targeting USP22 (75nM) for 48 hours, total protein was harvested and subjected to Western blot analysis. ***, P .001 vs control. C, The effects of USP22 depletion for 48 hours on Sirt1 expression. ***, P .001 vs control. D, The effects of USP22 depletion for 48 hours on Sirt1 ubiquitination. E, Immunofluorescence staining showed that USP22 was nuclear-localized protein in GMCs. Scale bars represented 20 m. F, GMCs were treated by AGEs (100 g/mL) for 12 hours to conduct Sirt1 coimmunoprecipitation reaction, the expression of Sirt1 and USP22 in Sirt1 immunoprecipitates and 10% WCL was detected by Western blot analysis. G, HEK-293T cells were cotransfected with Myc-Sirt1 and pRK5-HA-Ub, pRK5-HA-Ub K48, or pRK5-HA-Ub K63 for 48 hours. Then, Myc immunoprecipitation reaction was performed with anti-Myc antibody and immunoblotted with anti-HA antibody. H, USP22 deubiquitinated K48-linked ubiquitin chains on Sirt1 protein.

Article Snippet: PcDNA3-HA-Ub, pcDNA3-HA-Ub K0, vector pRK5, pRK5-HA-Ub, pRK5-HA-Ub K48, and pRK5-HA-Ub K63 were purchased from Addgene (http:// www.addgene.org/).

Techniques: Expressing, Transfection, Negative Control, Western Blot, Control, Ubiquitin Proteomics, Immunofluorescence, Staining, Immunoprecipitation

Journal: Oncotarget

Article Title: Sphingosine kinase 1 mediates AGEs-induced fibronectin upregulation in diabetic nephropathy

doi: 10.18632/oncotarget.20205

Figure Lengend Snippet: SphK1 was enriched by immunoprecipitation reaction in GMCs and subjected to western blot assay for detecting SphK1 ubiquitination (A) . Flag-SphK1 plasmid and pcDNA3-HA-Ub or pcDNA3-HA-Ub K0 plasmid was co-transfected in HEK-293A cells. After 3h of MG-132 treatment, cells were lysed to conduct immunoprecipitation reaction against SphK1 (B) . HEK-293A cells were co-transfected with Flag-SphK1 and vector pRK5, pRK5-HA-Ub, pRK5-HA-Ub K48, or pRK5-HA-Ub K63 for 48h. Then Flag immunoprecipitation reaction was performed with anti-Flag antibody and immunoblotted with anti-HA antibody (C) . CHX (1 μg/mL) was used to inhibit new protein synthesis in GMCs for detecting SphK1 stabilization (D) . ** P < 0.01, *** P < 0.001 vs. 0 h.

Article Snippet: PcDNA3-HA-Ub, pcDNA3-HA-Ub K0, vector pRK5, pRK5-HA-Ub, pRK5-HA-Ub K48, and pRK5-HA-Ub K63 were purchased from Addgene ( http://www.addgene.org/ ).

Techniques: Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Plasmid Preparation, Transfection

Journal: Journal of neurochemistry

Article Title: Degradation of gamma secretase activating protein by the ubiquitin-proteasome pathway

doi: 10.1111/jnc.13011

Figure Lengend Snippet: A. N2A-APPswe were cells transfected with empty vector or GSAP plasmid, then treated with the selective E3 UB ligase inhibitor SMER3 (15μM), the pan reversible inhibitor of deubiquitinases (DUBs) PR-619(10μM), and the proteasome inhibitors Z-IE (5μM) overnight. Upper panel. Cell lysates were immunoprecipitated (IP) with anti-GSAP antibody to pull down GSAP-associated proteins, the precipitates were then assayed by immunoblot analysis (IB) with anti-ubiquitin antibody to detect all the ubiquitinated GSAP. Lower panel. Total cell lysates from the condition described in A are directly assayed by Western blot (WB) with an antibody against ubiquitin. B. N2A-APPswe cells were transiently co-transfected with GSAP plus empty vector pRK5, or HA-tagged ubiquitin wilde type (WT), or HA-tagged ubiquitin mutant with only lysine 48 and mutation of all other lysines to arginines (K48), or HA-tagged ubiquitin mutant in which all lysines were mutated to arginines (KO). Upper panel. Cell lysates were first immunoprecipitated (IP) with anti-GSAP antibody to pull down GSAP-associated proteins, then the resultant immunoprecipated product was immunoblotted (IB) with an anti-HA antibody to detect all the ubiquitin in GSAP-containing immunoprecipitates. Lower panel. Total cell lysates from the condition described in B that are directly analyzed by Western Blot (WB) with anti-HA antibody and anti-GSAP antibody.

Article Snippet: For transfection, cells were grown to 70% confluence and separately transfected with 1 μg of empty vector pcDNA3.1 (Invitrogen, Carlsbad, CA), or GSAP cDNA (Origene, Rockville, MD), or empty vector pRK5 (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-WT (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-K48 (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-KO (Addgene, Cambridge MA), by using Lipofectamine® 2000 Transfection Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and as previously described ( 5 , 7 ).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: ASXL2 is monoubiquitinated on DEUBAD in a BAP1-dependent manner. a Schema representation of ASXLs protein family, Asx and BAP1/Calypso proteins. b Cartoon representation of BAP1/Ub/DEUBAD (DEUBAD of ASXL2) homology structure, based on the UCH37/Ub/DEUBAD (DEUBAD of RPN13) crystal structure (PDB, 4UEL). c BAP1 enhances ubiquitination of ASXL2. HEK293T cells were transfected with Myc-ASXL2, Flag-BAP1 or HA-Ub vectors and subjected to immunoprecipitation and immunoblotting. n = 3 biological replicates. d ASXL2 is monoubiquitinated in BAP1-dependent manner. HEK293T cells were transfected as indicated and ASXL2 ubiquitination with GFP-Ub was analyzed by immunoblotting. n = 4 biological replicates. e Decrease of ASXL2 protein levels in U-2 OS cells stably expressing shRNA of BAP1 . n = 5 biological replicates. f Depletion of ASXL2 using siRNA in U-2 OS cells. n = 5 biological replicates. g DEUBAD is required for ASXL2 monoubiquitination. HEK293T cells were co-transfected with Flag-BAP1 and the corresponding deletion mutants constructs of Myc-ASXL2 in presence or not of GFP-Ub and subjected to immunoblotting. n = 4 biological replicates. h The DEUBAD is sufficient for its monoubiquitination in BAP1-dependent manner. The indicated constructs were transfected in HEK293T cells and DEUBAD ubiquitination was analyzed. n = 4 biological replicates. i Mass spectrometry (MS) spectrum indicating Ub remnant on Lysine 370 of ASXL2. j Lysine 370 is the BAP1-dependent monoubiquitination site of ASXL2. HEK293T cells were transfected with the corresponding lysine mutants of DEUBAD (ASXL2) and used for immunoblotting. n = 3 biological replicates. k Conservation of the ASXLs ubiquitination site. Sequence alignment of ASXLs orthologs (ASXL2 K370 site highlighted in purple). l The DEUBAD of Asx is monoubiquitinated in Drosophila . S2 cells were transfected with Myc-V5-DEUBAD (Asx) and Myc-V5-DEUBAD (Asx) K325R expression vectors and subjected to immunoblotting. n = 3 biological replicates. m Drosophila DEUBAD K325 is monoubiquitinated in Calypso-dependent-manner. Myc-V5-DEUBAD (Asx) was co-transfected with control or Calypso dsRNA in S2 cells and its monoubiquitination levels were determined by immunoblotting. n = 3 biological replicates. Tubulin was used as a loading control for panels c – h and j . Histone H3 was used as a loading control for panels l , m

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Transfection, Immunoprecipitation, Western Blot, Stable Transfection, Expressing, shRNA, Construct, Mass Spectrometry, Sequencing

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: Monoubiquitination of DEUBAD requires BAP1 intramolecular interactions. a Representation of the different BAP1 mutant forms used for experiments done in the panels b and d . b Disruption of the intra-molecular interactions between the catalytic (UCH) and non-catalytic (CC1 and CTD) domains of BAP1 as well as inhibition of its interaction with DEUBAD by the R666-H669 cancer associated mutation impair DEUBAD domain monoubiquitination. HEK293T cells were transfected with BAP1 and its mutant forms as indicated in presence of Myc-DEUBAD (ASXL2) and subjected to western blotting. n = 4 biological replicates. c Reconstitution of the intramolecular interactions of BAP1 promotes DEUBAD monoubiquitination. Co-transfection of Myc-DEUBAD (ASXL2) or Myc-DEUBAD (ASXL2) K370R with the corresponding GFP BAP1- or Myc-BAP1- fragments fusion constructs in HEK293T and cell extracts were used for western blotting. The star indicates a possible degradation product. The band upper UCH or UCH-CC1 represents a potential post-translational modification of this domain. n = 2 biological replicates. d Monoubiquitination of the DEUBAD domain following expression of BAP1 ∆HBM , BAP1 ∆UCH , or BAP1 catalytic dead (C91S) constructs in HEK293T cells. Increased amounts of the different Flag-BAP1 constructs were used in presence of HA-Ub and Myc-DEUBAD (ASXL2), then DEUBAD monoubiquitination was visualized by western blotting. n = 4 biological replicates. e Validation of Ub binding mutants of BAP1. Flag-BAP1 and its different mutant forms were transfected in HEK293T cells. Cell lysates were labeled with HA-Ub-VME DUB probe and analyzed by immunoblotting. n = 2 biological replicates. f BAP1 ubiquitin binding is not required for DEUBAD monoubiquitination. HEK293T cells were transfected with BAP1 Ub binding mutants in presence of Myc-DEUBAD (ASXL2) and harvested for immunoblotting. n = 2 biological replicates. Tubulin was used as a loading control for panels b – f

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Mutagenesis, Inhibition, Transfection, Western Blot, Cotransfection, Construct, Modification, Expressing, Binding Assay, Labeling

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: Dual role of K370 ubiquitination on ASXL2 protein stability. a ASXL2 protein levels are accumulated following proteasome inhibition. U-2 OS or HEK293T cells were treated with 20 μM of MG132 for the indicated times and harvested for immunoblotting. n = 2 biological replicates. b HEK293T cells were treated with 20 μg/ml of cycloheximide (CHX) as indicated and ASXL2 levels were determined by immunoblotting. The arrows in panels a and b indicate the unmodified and the monoubiquitination forms of ASXL2. n = 2 biological replicates. c The non-modified form of ASXL2 is rapidly accumulated relative to its monoubiquitinated form following proteasome inhibition. U-2 OS cells were infected with lentiviral expressing vectors for ASXL2 or ASXL2 K370R then treated with 20 μM of MG132 for the indicated times and used for immunoblotting. n = 2 biological replicates. d Monoubiquitination of ASXL2 and mutation of K370 maintains its stability. U-2 OS cells stably expressing HA-ASXL2 or HA-ASXL2 K370R were treated with CHX as indicated and analyzed by immunoblotting as in c . n = 2 biological replicates. The arrows in panels c and d indicate the unmodified and the monoubiquitination forms of HA-ASXL2. e Monoubiquitination of DEUBAD and mutation of its K370 maintain its stability. U-2 OS cells stably expressing GFP-DEUBAD (ASXL2) or GFP-DEUBAD (ASXL2) K370R were treated with CHX as indicated. Cell lysates were used for immunoblotting. n = 3 biological replicates. f Unmodified DEUBAD is strongly accumulated relative to the monoubiquitinated form, following proteasome inhibition. U-2 OS cells stably expressing GFP-DEUBAD (ASXL2) or GFP-DEUBAD (ASXL2) K370R were treated with 20 μM of MG132 for the indicated times. Cell extracts were analyzed by immunoblotting. n = 3 biological replicates. g Further evidence that DEUBAD (ASXL2) K370 is also a site for Ub chain extension and degradation. HEK293T cells were transfected with Myc-DEUBAD (ASXL2) and HA-tagged Ub vectors and harvested for immunoblotting. n = 2 biological replicates. h ASXL2 stability is regulated by polyubiquitination inducing its degradation. HEK293T cells were transfected with Myc-ASXL2 and HA-tagged Ub vectors as indicated and subjected to immunoblotting. n = 2 biological replicates. Tubulin was used as a loading control for all panels

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Inhibition, Western Blot, Modification, Infection, Expressing, Mutagenesis, Stable Transfection, Transfection

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: K370 is the site of mono- and polyubiquitination. a DEUBAD protein levels are affected by DUB depletion. U-2 OS cells stably expressing Flag-HA-BAP1 and GFP-DEUBAD (ASXL2) were transfected with siRNA library for all human DUBs (see also Supplementary Fig. ). Densitometry analysis of protein bands in DUB siRNA versus non-target (NT) siRNA control were conducted. The arrows indicate the top DUBs hits that increased the corresponding protein levels. n = 1 biological sample. b Comparison of changes in band intensity between unmodified and monoubiquitinated forms of DEUBAD (ASXL2). c Validation that K370 is the site of mono- and poly-ubiquitination of DEUBAD (ASXL2). U-2 OS cells stably expressing Flag-HA-BAP1 and either GFP-DEUBAD (ASXL2) or GFP-DEUBAD (ASXL2) K370R were transfected with siRNA NT control or siRNAs targeting different DUBs and harvested for western blotting as indicated. n = 2 biological replicates. Tubulin was used as a loading control for panel c

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: DEUBAD monoubiquitination is directly catalyzed by UBE2E family. a UBE2E1 and UBE2E3 directly catalyze monoubiquitination of DEUBAD (ASXL2). Bacterial purified His-BAP1/MBP-DEUBAD (ASXL2) complex was incubated with the indicated recombinant UBE2s conjugating enzymes for in vitro ubiquitination assays. The reactions were analyzed by western blot as indicated. The black dots indicate UBE2E1 and UBE2E3. n = 1 biological sample. b UBE2E3 in vitro monoubiquitinates K370 of DEUBAD (ASXL2) alone or DEUBAD (ASXL2) in complex with BAP1. Bacteria purified MBP-DEUBAD (ASXL2), MBP-DEUBAD (ASXL2) K370R, His-BAP1/MBP-DEUBAD (ASXL2) and His-BAP1/MBP-DEUBAD (ASXL2) K370R complexes were used for in vitro ubiquitination assays with bacteria-purified UBE2E3 and analyzed by immunoblotting. The arrows indicate the monoubiquitinated form of DEUBAD domain. n = 3 biological replicates. c Bacteria purified His-BAP1/MBP-DEUBAD (ASXL2) or His-BAP1 C91S/MBP-DEUBAD (ASXL2) complexes were used for in vitro ubiquitination assays. Reactions were stopped at the indicated times for immunoblotting. d In vitro ubiquitination assays were conducted using bacteria purified His-BAP1/MBP-DEUBAD (ASXL2) complex or MBP-DEUBAD (ASXL2) in complex with the corresponding BAP1 mutants. Reactions were done for the indicted times and analyzed by immunoblotting. n = 2 biological replicates for panels c and d

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Purification, Incubation, Recombinant, In Vitro, Western Blot

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: Dual role of UBE2E family in regulating ASXL2 protein stability. a Depletion of UBE2E3 results in increased DEUBAD (ASXL2) protein levels. U-2 OS cells stably expressing GFP-DEUBAD (ASXL2) were transfected with a different siRNAs oligos for UBE2E3 and then treated with CHX as indicated. Right panel, densitometry analysis of DEUBAD protein bands was conducted and presented as indicated. n = 3 biological replicates. b Combined depletion of all three UBE2Es resulted in a stronger increase of DEUBAD (ASXL2) protein levels which become similar to those of GFP-DEUBAD K370R. U-2 OS cells stably expressing GFP-DEUBAD (ASXL2) were transfected with Non-target siRNA control or a combination of UBE2Es ( UBE2E1 , UBE2E2 , UBE2E3 ) siRNAs, treated with CHX as indicated and used for immunoblotting. Right panel, densitometry analysis of the protein levels of DEUBAD bands were conducted and presented as in a . n = 3 biological replicates. c Opposite effects of UBE2Es on DEUBAD (ASXL2) and ASXL2 protein levels depending on BAP1 expression. Flag-UBE2Es expression constructs were transfected in HEK293T cells in the presence of Myc-DEUBAD (ASXL2) or Myc-ASXL2 expression vectors in presence or not of BAP1 and cells were harvested for immunoblotting. n = 3 biological replicates. d Depletion of UBE2E1, UBE2E3 and the combination of three UBE2Es resulted in decreased protein levels of endogenous ASXL2 and BAP1. U-2 OS cells were transfected with Non-target control (NT) or UBE2E siRNAs and then treated with CHX and used for immunoblotting. Bottom panel, densitometry analysis of ASXL2 and BAP1 levels was conducted and presented for each siRNA. n = 3 biological replicates. e UBE2Es-mediated DEUBAD monoubiquitination is conserved in Drosophila . S2 cells were transfected with dsRNA for Calypso or UBCD2 and Myc-V5-DEUBAD (Asx). DEUBAD monoubiquitination was evaluated by immunoblotting. Bottom panel, densitometry analysis of the protein levels of monoubiquitinated form versus non-modified form of DEUBAD (Asx). n = 3 biological replicates. The stars in panels a , b , and d indicate UBE2E2 band detected with UBE2E1 antibody. Tubulin was used as a loading control for panels a – d and histone H3 was used as a loading control for panel e . Error bars in panels a , b , d , e represents s.d. (mean ± SD)

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Construct, Modification

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: Monoubiquitination of DEUBAD K370 promotes BAP1 DUB activity. a Expression of DEUBAD (ASXL2) but not DEUBAD (ASXL2) K370R, results in reduced levels of H2Aub in BAP1-dependent manner. U-2 OS cells stably expressing empty vector or Flag-HA-BAP1 were transduced with lentiviral expressing vectors for either GFP-DEUBAD (ASXL2) or GFP-DEUBAD2 (ASXL2) K370R and endogenous level of H2Aub was analyzed by immunofluorescence. GFP DEUBAD (ASXL2) (green), H2Aub (red), DAPI (blue). n = 3 biological replicates. b DEUBAD induces reduction of H2Aub levels in BAP1 catalytic activity dependent manner. U-2 OS cells stably expressing Flag-HA-BAP1 C91S were infected with either GFP-DEUBAD (ASXL2) or GFP-DEUBAD (ASXL2) K370R and H2Aub levels were assessed (see also Supplementary Fig. ). n = 2 biological replicates. c Expression of ASXL2, but not ASXL2 K370R, results in reduced levels of H2Aub. U-2 OS cells stably expressing Flag-HA-BAP1 were infected with lentiviral expressing vectors for either Myc-ASXL2 or Myc-ASXL2 K370R and H2Aub changes were evaluated by immunostaining. n = 2 biological replicates. The cells showing either decrease or no change of H2Aub levels were encircled in panels a – c . Scale bar: 10 μm for panels a – c . d Monoubiquitinated form of DEUBAD (ASXL2) strongly promotes BAP1 DUB activity comparatively to DEUBAD (ASXL2) K370R. Purified BAP1/DEUBAD complexes from HEK293T cells were used for in vitro DUB assays with Flag-H2A nucleosomes and analyzed by immunoblotting. n = 2 biological replicates. e Monoubiquitinated DEUBAD promotes deubiquitination of H2Aub in Drosophila . S2 cells were transfected with either Myc-V5-DEUBAD (Asx) or Myc-V5-DEUBAD (Asx) K325R and harvested for immunoblotting. Right panel, densitometry analysis of the levels of H2Aub is shown. Error bars represent s.d. (mean ± SD). n = 3 biological replicates. Histone H3 was used as a loading control for panels d and e

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Activity Assay, Expressing, Stable Transfection, Plasmid Preparation, Transduction, Immunofluorescence, Infection, Immunostaining, Purification, In Vitro, Western Blot, Transfection

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: Expression of ASXL2 K370R reduces mammalian cell proliferation. a Enforced expression of ASXL2 K370R decreases cellular proliferation. U-2 OS cells were transduced with different amounts of lentiviral suspensions produced using ASXL2 or ASXL2 K370R constructs. Cells were selected by puromycin and harvested for immunoblotting (top panel). Equal numbers of puromycin-selected cells were plated for colony formation assay (CFA) (bottom panel). n = 2 biological replicates. b The cells infected in a , were treated with nocodazole for FACS analysis at the indicated times. Note that (+2×) refers to transduction of cells with twice the amount of virus we normally use for Myc-ASXL2, and (−2×) refers to transduction of the cells with two times less the amount of viruses we normally use for ASXL2 370R. This adjustment was conducted to correct for the expression levels usually higher for ASXL2 K370R. n = 2 biological replicates. c , d Normal diploid fibroblast IMR90 cells were transduced with viral expression constructs for ASXL2 or ASXL2 K370R. Cells were selected by puromycin and equal numbers were plated for phase contrast pictures ( c ) or cell counts ( d ). Scale bar: 50 µm for panel c . n = 2 biological replicates. (Exp.1 and Exp.2). e siRNA depletion of ASXL2 decreases cellular proliferation. U-2 OS cells were transfected with NT siRNA control or siRNA for ASXL2 . Equal numbers of puromycin-selected cells were plated for CFA (left panel). Cells were treated with nocodazole for FACS analysis (right panel). f siRNA depletion of UBE2E3 decreases cellular proliferation. U-2 OS cells were transfected with individual siRNA constructs as indicated. Cells were plated for viability measurement using MTT assay. n = 3 biological replicates. Error bars represent s.d. (mean ± SD). g , h Inactivation of UBE2E3 locus decreases cellular proliferation. Schematic representation for gRNAs targeting the UBE2E3 locus ( g top panel). U-2 OS cells were transduced with different lentiviral CRISPR/Cas9 constructs, selected by puromycin and harvested for immunoblotting ( g bottom panel). n = 3 biological replicates. Equal numbers of puromycin-selected cells were plated for CFA ( h ). n = 2 biological replicates. i The cells selected as in h were treated with nocodazole for FACS analysis at the indicated time . n = 2 biological replicates. j Positive correlation of BAP1, ASXL2, and UBE2E3 protein expression levels in human mesothelioma. Mesothelioma biopsies were immunostained for ASXL2, UBE2E3, or BAP1 (see Supplementary Fig. and Supplementary Table ). Pictures were taken at 100× magnification. Scale bar: 100 μm. Tubulin was used as a loading control for panels a and g

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Expressing, Transduction, Produced, Construct, Western Blot, Colony Assay, Infection, Transfection, MTT Assay, CRISPR

Journal: Nature Communications

Article Title: Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1

doi: 10.1038/s41467-018-06854-2

Figure Lengend Snippet: Regulation of the BAP1/ASXL2 complexes by DEUBAD ubiquitination. ASXL2 is constitutively monoubiquitinated by UBE2Es on its DEUBAD domain. Interaction of monoubiquitinated ASXL2 with BAP1 leads to its stabilization and the subsequent activation of the DUB complex. Otherwise monoubiquitinated ASXL2 is targeted by other E3 Ub-ligases for ubiquitin chain extension and proteasomal degradation. UBE2Es also target ASXL2 already in complex with BAP1, possibly dynamically regulating its activity. DUBs might also regulate the stability of both free and complexed ASXL2. Mutation of ASXL2 lysine 370 or cancer mutations that abolish ASXL2-BAP1 interaction lead to defective monoubiquitination and subsequent loss of BAP1 DUB activity and tumor suppression

Article Snippet: 3xFlag-DEUBAD (ASXL2) WT and 3xFlag-2HA-DEUBAD (ASXL2) K370R were generated by subcloning of DEUBAD (ASXL2) WT and DEUBAD (ASXL2) K370R cDNA into pLPC vector. pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K48R, pRK5-HA-Ubiquitin-K63, pRK5-HA-Ubiquitin-K0, and pCDNA-HA-UCH37 were purchased from Addgene (#17605, #17604, #17606, #17603, and # 19415).

Techniques: Activation Assay, Activity Assay, Mutagenesis