his ube2es  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Name:
    Amylose Resin
    Description:
    Amylose Resin 100 ml
    Catalog Number:
    e8021l
    Price:
    1158
    Size:
    100 ml
    Category:
    Protein Purification Kit Components
    Buy from Supplier


    Structured Review

    New England Biolabs his ube2es
    Amylose Resin
    Amylose Resin 100 ml
    https://www.bioz.com/result/his ube2es/product/New England Biolabs
    Average 92 stars, based on 1261 article reviews
    Price from $9.99 to $1999.99
    his ube2es - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Activity Assay, Mutagenesis

    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)
    Figure Legend 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)

    Techniques Used: Stable Transfection, Expressing, Transfection, Construct, Modification

    2) Product Images from "Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase"

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase

    Journal: International journal for parasitology

    doi: 10.1016/j.ijpara.2006.10.014

    SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach (amylose resin-based affinity chromatography and PAGE gel extraction). The full-length fusion protein (138-kDa) was used in all enzymatic assays. M = protein marker, lane 1 = full length fusion protein from gel purification, lane 2 = amylose resin-based affinity purified protein.
    Figure Legend Snippet: SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach (amylose resin-based affinity chromatography and PAGE gel extraction). The full-length fusion protein (138-kDa) was used in all enzymatic assays. M = protein marker, lane 1 = full length fusion protein from gel purification, lane 2 = amylose resin-based affinity purified protein.

    Techniques Used: SDS Page, Binding Assay, Purification, Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Gel Extraction, Marker, Gel Purification, Affinity Purification

    3) Product Images from "An unfolded protein-induced conformational switch activates mammalian IRE1"

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1

    Journal: eLife

    doi: 10.7554/eLife.30700

    hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p
    Figure Legend Snippet: hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p

    Techniques Used: Binding Assay

    4) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    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
    Figure Legend 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

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

    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. 7 ). 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
    Figure Legend 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. 7 ). 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

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

    5) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Activity Assay, Mutagenesis

    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
    Figure Legend 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

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

    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)
    Figure Legend 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)

    Techniques Used: Stable Transfection, Expressing, Transfection, Construct, Modification

    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 ). 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
    Figure Legend 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 ). 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

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

    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
    Figure Legend 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

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

    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
    Figure Legend 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

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

    K370 is the site of mono- and polyubiquitination. a ). 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
    Figure Legend Snippet: K370 is the site of mono- and polyubiquitination. a ). 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

    Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot

    6) Product Images from "Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation"

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201609073

    Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.
    Figure Legend Snippet: Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

    Techniques Used: In Vivo, In Vitro, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Immunofluorescence, Staining, Construct, Functional Assay, Mutagenesis

    7) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    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
    Figure Legend 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

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

    8) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Activity Assay, Mutagenesis

    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. 11a and Supplementary Table 3 ). Pictures were taken at 100× magnification. Scale bar: 100 μm. Tubulin was used as a loading control for panels a and g
    Figure Legend 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. 11a and Supplementary Table 3 ). Pictures were taken at 100× magnification. Scale bar: 100 μm. Tubulin was used as a loading control for panels a and g

    Techniques Used: Expressing, Transduction, Produced, Construct, Colony Assay, Infection, FACS, Transfection, MTT Assay, CRISPR

    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
    Figure Legend 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

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

    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)
    Figure Legend 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)

    Techniques Used: Stable Transfection, Expressing, Transfection, Construct, Modification

    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. 7 ). 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
    Figure Legend 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. 7 ). 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

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

    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
    Figure Legend 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

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

    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
    Figure Legend 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

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

    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. 3b ). 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
    Figure Legend 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. 3b ). 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

    Techniques Used: Stable Transfection, Expressing, Transfection, Western Blot

    9) Product Images from "Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *"

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.525279

    UNC1215 blocks DNMT1K142me1 MBT domain interaction. A , UNC1215 inhibits the binding of biotin-DNMT1K142me1 peptide to recombinant PHF20L1a in vitro . Constant amounts of recombinant MBP-PHF20L1a and biotin-tagged DNMT1K142me1 peptide prebound to streptavidin beads with increasing concentrations of UNC1215 are indicated at the top . Equal loading of streptavidin was illustrated by Ponceau staining, whereas anti-MBP antibody was used to reveal MBP-PHF20L1a. Densitometry measurements of MBP-PHF20L1a, normalized to streptavidin, are shown at the bottom and illustrate the percentage of bound MBP-PHF20L1. B , UNC1215 treatment decreases levels of DNMT1 in HeLa cells. Cells were treated with increasing concentrations of UNC1215 (indicated at the top), and extracts were analyzed by Western blot (antibodies indicated to the right ). Densitometry measurements illustrating the percentage of DNMT1 protein levels, normalized to β-actin, are shown in the middle panel . Real-time quantitative PCR measurement of DNMT1 mRNA in response to UNC1215 treatment is shown in the far right panel. C , UNC1215 treatment of HeLa cells reduces in vivo binding of PHF20L1 to DNMT1. DNMT1 immunoprecipitations ( IP ) were performed on HeLa cells treated with either 80 μ m UNC1215 (+) or ethanol (−). Western blot antibodies for DNMT1 or PHF20L1 were then used to visualize the DNMT1 immunoprecipitation. Error bars , S.D.
    Figure Legend Snippet: UNC1215 blocks DNMT1K142me1 MBT domain interaction. A , UNC1215 inhibits the binding of biotin-DNMT1K142me1 peptide to recombinant PHF20L1a in vitro . Constant amounts of recombinant MBP-PHF20L1a and biotin-tagged DNMT1K142me1 peptide prebound to streptavidin beads with increasing concentrations of UNC1215 are indicated at the top . Equal loading of streptavidin was illustrated by Ponceau staining, whereas anti-MBP antibody was used to reveal MBP-PHF20L1a. Densitometry measurements of MBP-PHF20L1a, normalized to streptavidin, are shown at the bottom and illustrate the percentage of bound MBP-PHF20L1. B , UNC1215 treatment decreases levels of DNMT1 in HeLa cells. Cells were treated with increasing concentrations of UNC1215 (indicated at the top), and extracts were analyzed by Western blot (antibodies indicated to the right ). Densitometry measurements illustrating the percentage of DNMT1 protein levels, normalized to β-actin, are shown in the middle panel . Real-time quantitative PCR measurement of DNMT1 mRNA in response to UNC1215 treatment is shown in the far right panel. C , UNC1215 treatment of HeLa cells reduces in vivo binding of PHF20L1 to DNMT1. DNMT1 immunoprecipitations ( IP ) were performed on HeLa cells treated with either 80 μ m UNC1215 (+) or ethanol (−). Western blot antibodies for DNMT1 or PHF20L1 were then used to visualize the DNMT1 immunoprecipitation. Error bars , S.D.

    Techniques Used: Binding Assay, Recombinant, In Vitro, Staining, Western Blot, Real-time Polymerase Chain Reaction, In Vivo, Immunoprecipitation

    10) Product Images from "Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1"

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

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06854-2

    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
    Figure Legend 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

    Techniques Used: Activation Assay, Activity Assay, Mutagenesis

    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)
    Figure Legend 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)

    Techniques Used: Stable Transfection, Expressing, Transfection, Construct, Modification

    11) Product Images from "Stable MOB1 interaction with Hippo/MST is not essential for development and tissue growth control"

    Article Title: Stable MOB1 interaction with Hippo/MST is not essential for development and tissue growth control

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00795-y

    The crystal structure of the MOB1/NDR2 complex reveals phosphorylation-independent interactions of MOB1 with NDR2 through conserved interfaces. a Overall crystal structure of the complex between human MOB1A (residues 33–216) and human NDR2 (residues 25–88). MOB1 and NDR2 are colored in magenta and green , respectively. The Zinc associated with MOB1 is indicated in gray . Secondary structure elements are highlighted. b The negatively charged surface of MOB1 recognizes positively charged residues on NDR2. The electrostatic surface potential of MOB1 is shown ( red : negatively charged residues; blue : positively charged residues). NDR2 is shown as a ribbon representation colored in green . c The MOB1/NDR2 binding surfaces are highly conserved. The residues of MOB1 are gradually colored according to conservation scores ( dark red : most conserved residues; dark cyan : least conserved residues). NDR2 is shown as a ribbon representation colored in green . d Structure based sequence alignment of MOB1 from indicated species. MOB1 residues mediating interactions with NDR2 are marked with brown (main-chain hydrogen bonds), green (side-chain hydrogen bonds), and magenta circles (van der Waals interactions). α helices are painted with cyan background. Thr12 and Thr35 phosphorylation sites are indicated by red arrows . e , f The interaction interfaces of MOB1 with the α1 (residues 25–56) and α2 (residues 61–84) helices of NDR2. MOB1 and NDR2 and their key interacting residues are shown in pink and green , respectively. Dashed lines represent hydrogen bonds. Side-chain nitrogen, oxygen, and sulfur atoms of MOB1 and NDR2 residues are colored in blue , red and gold , respectively. The hydrogen bond network connecting the α1 and α2 helices of NDR2 is also illustrated in f . For a stereo image of a portion of the electron density map see Supplementary Fig. 18
    Figure Legend Snippet: The crystal structure of the MOB1/NDR2 complex reveals phosphorylation-independent interactions of MOB1 with NDR2 through conserved interfaces. a Overall crystal structure of the complex between human MOB1A (residues 33–216) and human NDR2 (residues 25–88). MOB1 and NDR2 are colored in magenta and green , respectively. The Zinc associated with MOB1 is indicated in gray . Secondary structure elements are highlighted. b The negatively charged surface of MOB1 recognizes positively charged residues on NDR2. The electrostatic surface potential of MOB1 is shown ( red : negatively charged residues; blue : positively charged residues). NDR2 is shown as a ribbon representation colored in green . c The MOB1/NDR2 binding surfaces are highly conserved. The residues of MOB1 are gradually colored according to conservation scores ( dark red : most conserved residues; dark cyan : least conserved residues). NDR2 is shown as a ribbon representation colored in green . d Structure based sequence alignment of MOB1 from indicated species. MOB1 residues mediating interactions with NDR2 are marked with brown (main-chain hydrogen bonds), green (side-chain hydrogen bonds), and magenta circles (van der Waals interactions). α helices are painted with cyan background. Thr12 and Thr35 phosphorylation sites are indicated by red arrows . e , f The interaction interfaces of MOB1 with the α1 (residues 25–56) and α2 (residues 61–84) helices of NDR2. MOB1 and NDR2 and their key interacting residues are shown in pink and green , respectively. Dashed lines represent hydrogen bonds. Side-chain nitrogen, oxygen, and sulfur atoms of MOB1 and NDR2 residues are colored in blue , red and gold , respectively. The hydrogen bond network connecting the α1 and α2 helices of NDR2 is also illustrated in f . For a stereo image of a portion of the electron density map see Supplementary Fig. 18

    Techniques Used: Binding Assay, Sequencing

    MOB1 binds differently to the NTR domains of NDR2 and LATS1 through key residues. a Structure based sequence alignment of the conserved N-terminal regulatory domain of NDR/LATS kinases that is required for MOB1 binding. NDR2 residues mediating interactions with MOB1 are marked with red circles . Residues mediating intramolecular interactions of NDR2 are denoted by blue triangles . Residues conserved from yeast to humans are highlighted in green , while residues conserved in at least seven of the conserved NDR/LATS family members are marked in cyan . α helices are indicated with gray rods . b Asp63 of MOB1 is involved in LATS1 binding, but does not contribute to the interaction of MOB1 with NDR2. MOB1 is shown in yellow , while NDR2 and LATS1 are indicated in green and cyan , respectively. Top panel , Phe31 of NDR2 does not interact with Asp63 of MOB1. Bottom panel , His646 of LATS1 forms a hydrogen bond with Asp63 of MOB1. c – f Isothermal titration calorimetry ( ITC ) assays measuring the dissociation constant ( K d ) of indicated non-phosphorylated full-length MOB1 or MST2-phosphorylated MOB1 (phospho-MOB1) with wild-type and mutant NDR2 (25–88) or LATS1 (640–703) variants. MOB1/NDR2 complex formation was dramatically increased by prior phosphorylation of MOB1 c . ITC measurements could not detect any interaction between LATS1 wild-type and non-phosphorylated MOB1, while phospho-MOB1 bound to LATS1 d . The NDR2(Y32V) mutant did not associate with non-phosphorylated MOB1, but bound to phospho-MOB1, although with a 20-fold decreased binding affinity compared to wild-type NDR2 ( e ). The mutation of Val647 to Tyr of LATS1 enabled LATS1 to bind to non-phosphorylated MOB1, and LATS1(V647Y) displayed a 5-fold increased binding affinity for phospho-MOB1 compared to wild-type LATS1 ( f )
    Figure Legend Snippet: MOB1 binds differently to the NTR domains of NDR2 and LATS1 through key residues. a Structure based sequence alignment of the conserved N-terminal regulatory domain of NDR/LATS kinases that is required for MOB1 binding. NDR2 residues mediating interactions with MOB1 are marked with red circles . Residues mediating intramolecular interactions of NDR2 are denoted by blue triangles . Residues conserved from yeast to humans are highlighted in green , while residues conserved in at least seven of the conserved NDR/LATS family members are marked in cyan . α helices are indicated with gray rods . b Asp63 of MOB1 is involved in LATS1 binding, but does not contribute to the interaction of MOB1 with NDR2. MOB1 is shown in yellow , while NDR2 and LATS1 are indicated in green and cyan , respectively. Top panel , Phe31 of NDR2 does not interact with Asp63 of MOB1. Bottom panel , His646 of LATS1 forms a hydrogen bond with Asp63 of MOB1. c – f Isothermal titration calorimetry ( ITC ) assays measuring the dissociation constant ( K d ) of indicated non-phosphorylated full-length MOB1 or MST2-phosphorylated MOB1 (phospho-MOB1) with wild-type and mutant NDR2 (25–88) or LATS1 (640–703) variants. MOB1/NDR2 complex formation was dramatically increased by prior phosphorylation of MOB1 c . ITC measurements could not detect any interaction between LATS1 wild-type and non-phosphorylated MOB1, while phospho-MOB1 bound to LATS1 d . The NDR2(Y32V) mutant did not associate with non-phosphorylated MOB1, but bound to phospho-MOB1, although with a 20-fold decreased binding affinity compared to wild-type NDR2 ( e ). The mutation of Val647 to Tyr of LATS1 enabled LATS1 to bind to non-phosphorylated MOB1, and LATS1(V647Y) displayed a 5-fold increased binding affinity for phospho-MOB1 compared to wild-type LATS1 ( f )

    Techniques Used: Sequencing, Binding Assay, Isothermal Titration Calorimetry, Mutagenesis

    MOB1 binding to LATS1/2 is required to suppress anchorage-independent growth of human cancer cells, while MOB1 binding to MST1/2 is dispensable. a Immunoblotting with indicated antibodies of cell lysates derived from MCF-7 human breast cancer cells stably expressing the indicated HA-MOB1 variants or empty vector ( EV ) as negative control in lane 1. Relative molecular weights are indicated. See Supplementary Fig. 19 for uncropped western blots. b Proliferation rates of attached MCF-7 cells stably expressing indicated HA-MOB1A variants. The average of three experiments performed in triplicates with three independent cell pools is shown ( n = 3). Statistically significant differences between EV and all MOB1 variant expressing cell pools are indicated ( *p
    Figure Legend Snippet: MOB1 binding to LATS1/2 is required to suppress anchorage-independent growth of human cancer cells, while MOB1 binding to MST1/2 is dispensable. a Immunoblotting with indicated antibodies of cell lysates derived from MCF-7 human breast cancer cells stably expressing the indicated HA-MOB1 variants or empty vector ( EV ) as negative control in lane 1. Relative molecular weights are indicated. See Supplementary Fig. 19 for uncropped western blots. b Proliferation rates of attached MCF-7 cells stably expressing indicated HA-MOB1A variants. The average of three experiments performed in triplicates with three independent cell pools is shown ( n = 3). Statistically significant differences between EV and all MOB1 variant expressing cell pools are indicated ( *p

    Techniques Used: Binding Assay, Derivative Assay, Stable Transfection, Expressing, Plasmid Preparation, Negative Control, Western Blot, Variant Assay

    Definition of MOB1 variants with selective loss-of-interaction with Hippo core kinases. a Lysates of HEK293 cells expressing full-length HA-MST1 or HA-MST2 wild-type ( wt ) together with indicated full-length MOB1A versions were subjected to immunoprecipitation ( IP ) using anti-HA. Complexes were studied by immunoblotting using anti-myc ( top ) and anti-HA ( middle ). Input lysates were analyzed with anti-myc ( bottom ). The K104E/K105E mutations caused loss of binding to MST1/2. See Supplementary Fig. 19 for uncropped western blots. b Lysates of Drosophila S2R + cells expressing full-length HA-Hpo(wt) together with indicated full-length MOB1A versions were subjected to IP using anti-HA. Complexes were studied by immunoblotting using anti-myc ( top ) and anti-HA ( middle ). Input lysates were probed with anti-myc ( bottom ). Relative molecular weights are shown. See Supplementary Fig. 19 for uncropped western blots. c Schematic summary of the biochemical and molecular characterization of the indicated MOB1A variants presented in Supplementary Figs. 5 – 13 . Noteworthy, NDR2 weakly interacted with MOB1(E51K), and Warts and Hpo bound normally to the E51K mutant as judged by co-immunoprecipitation experiments (marked by an asterisk ), hence the E51K mutant was not studied further. d Model of human MOB1A(33–216) depicting the possibly opposing binding sites on MOB1 of NDR/LATS and MST1/2 kinases. Secondary structure elements of MOB1 are highlighted. The locations of Glu51, Asp63, and Lys104/Lys105 in MOB1 are indicated
    Figure Legend Snippet: Definition of MOB1 variants with selective loss-of-interaction with Hippo core kinases. a Lysates of HEK293 cells expressing full-length HA-MST1 or HA-MST2 wild-type ( wt ) together with indicated full-length MOB1A versions were subjected to immunoprecipitation ( IP ) using anti-HA. Complexes were studied by immunoblotting using anti-myc ( top ) and anti-HA ( middle ). Input lysates were analyzed with anti-myc ( bottom ). The K104E/K105E mutations caused loss of binding to MST1/2. See Supplementary Fig. 19 for uncropped western blots. b Lysates of Drosophila S2R + cells expressing full-length HA-Hpo(wt) together with indicated full-length MOB1A versions were subjected to IP using anti-HA. Complexes were studied by immunoblotting using anti-myc ( top ) and anti-HA ( middle ). Input lysates were probed with anti-myc ( bottom ). Relative molecular weights are shown. See Supplementary Fig. 19 for uncropped western blots. c Schematic summary of the biochemical and molecular characterization of the indicated MOB1A variants presented in Supplementary Figs. 5 – 13 . Noteworthy, NDR2 weakly interacted with MOB1(E51K), and Warts and Hpo bound normally to the E51K mutant as judged by co-immunoprecipitation experiments (marked by an asterisk ), hence the E51K mutant was not studied further. d Model of human MOB1A(33–216) depicting the possibly opposing binding sites on MOB1 of NDR/LATS and MST1/2 kinases. Secondary structure elements of MOB1 are highlighted. The locations of Glu51, Asp63, and Lys104/Lys105 in MOB1 are indicated

    Techniques Used: Expressing, Immunoprecipitation, Binding Assay, Western Blot, Mutagenesis

    12) Product Images from "Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein"

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008381

    On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.
    Figure Legend Snippet: On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.

    Techniques Used: Copurification, Labeling, SDS Page, Staining, Fluorescence, Expressing, Incubation, Purification

    Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.
    Figure Legend Snippet: Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.

    Techniques Used: Labeling, Binding Assay, Purification, Sequencing, Incubation, SDS Page, Staining, Western Blot, Fluorescence, Affinity Chromatography

    13) Product Images from "LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1"

    Article Title: LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1

    Journal: Oncogenesis

    doi: 10.1038/s41389-019-0182-7

    Identification of PTBP1 as a MACC1-AS1-binding partner. a PTBP1 co-precipitated with MACC1-AS1. Upper: MACC1-AS1-MS2-pulldown assays, MS2 was used as a control. Lower: western blot analysis of the presence of PTBP1 in the MACC1-AS1-MS2 precipitates. b MACC1-AS1 was detected in the PTBP1 IP. Upper: western blots of PTBP1 in the IP, normal IgG was used as a control. Lower: RT-PCR and agarose gel electrophoresis indicated the association of MACC1-AS1 with PTBP1. c Schematic representation of truncated MACC1-AS1-MS2 RNAs, which can be bound to amylose resin through MBP-MCP. d Upper: vectors expressing MS2-fused full-length MACC1-AS1 or truncated MACC1-AS1 (5′, middle (m) and 3′) were transiently transfected into HEK-293T cells. RT-PCR showing that individual MS2-fused RNAs were pulled down by MBP-MCP attached amylose resin. Lower: western blots indicating that PTBP1 co-precipitated with the full-length and the 3′ part of MACC1-AS1. e Upper: a putative motif for the PTBP1 binding site within MACC1-AS1 is shown. Red nucleotides indicates the mutated sequences. Lower; wild-type and mutant MACC1-AS1-MS2 RNAs were transfected into HEK-293T cells. MS2-pulldown assays and immunoblots showed that PTBP1 was barely detected in the PTBP1 precipitates when the PTBP1-binding motif within MACC1-AS1 was mutated.
    Figure Legend Snippet: Identification of PTBP1 as a MACC1-AS1-binding partner. a PTBP1 co-precipitated with MACC1-AS1. Upper: MACC1-AS1-MS2-pulldown assays, MS2 was used as a control. Lower: western blot analysis of the presence of PTBP1 in the MACC1-AS1-MS2 precipitates. b MACC1-AS1 was detected in the PTBP1 IP. Upper: western blots of PTBP1 in the IP, normal IgG was used as a control. Lower: RT-PCR and agarose gel electrophoresis indicated the association of MACC1-AS1 with PTBP1. c Schematic representation of truncated MACC1-AS1-MS2 RNAs, which can be bound to amylose resin through MBP-MCP. d Upper: vectors expressing MS2-fused full-length MACC1-AS1 or truncated MACC1-AS1 (5′, middle (m) and 3′) were transiently transfected into HEK-293T cells. RT-PCR showing that individual MS2-fused RNAs were pulled down by MBP-MCP attached amylose resin. Lower: western blots indicating that PTBP1 co-precipitated with the full-length and the 3′ part of MACC1-AS1. e Upper: a putative motif for the PTBP1 binding site within MACC1-AS1 is shown. Red nucleotides indicates the mutated sequences. Lower; wild-type and mutant MACC1-AS1-MS2 RNAs were transfected into HEK-293T cells. MS2-pulldown assays and immunoblots showed that PTBP1 was barely detected in the PTBP1 precipitates when the PTBP1-binding motif within MACC1-AS1 was mutated.

    Techniques Used: Binding Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Transfection, Mutagenesis

    Role of MACC1-AS1 on MACC1 mRNA expression. a , b qRT-PCR showed that levels of MACC1 mRNA were increased in MACC1-AS1-overexpressing MCF7 and MDA-MB-231 cells. c Upper: schematic representation of the MACC1-AS1-MS2 chimeric RNA, which can be bound to amylose resin through recombinant protein MBP-MCP. Lower: RT-PCR and agarose gel electrophoresis showing that MS2-tagged MACC1-AS1 RNA was pulled down by MBP-MCP. d qRT-PCR showed that MACC1 mRNA was not detected in the precipitates of MACC1-AS1. e Representative western blots showing the expression of α-tubulin and fibrillarin in cytosolic ( c ) and nuclear (N) fractions of MACC1-AS1-overexpressing MDA-MB-231 cells. f qRT-PCR indicated that MACC1-AS1 is mainly present in the cytosolic fraction.
    Figure Legend Snippet: Role of MACC1-AS1 on MACC1 mRNA expression. a , b qRT-PCR showed that levels of MACC1 mRNA were increased in MACC1-AS1-overexpressing MCF7 and MDA-MB-231 cells. c Upper: schematic representation of the MACC1-AS1-MS2 chimeric RNA, which can be bound to amylose resin through recombinant protein MBP-MCP. Lower: RT-PCR and agarose gel electrophoresis showing that MS2-tagged MACC1-AS1 RNA was pulled down by MBP-MCP. d qRT-PCR showed that MACC1 mRNA was not detected in the precipitates of MACC1-AS1. e Representative western blots showing the expression of α-tubulin and fibrillarin in cytosolic ( c ) and nuclear (N) fractions of MACC1-AS1-overexpressing MDA-MB-231 cells. f qRT-PCR indicated that MACC1-AS1 is mainly present in the cytosolic fraction.

    Techniques Used: Expressing, Quantitative RT-PCR, Multiple Displacement Amplification, Recombinant, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot

    14) Product Images from "PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands"

    Article Title: PDZ Affinity Chromatography: A general method for affinity purification of proteins based on PDZ domains and their ligands

    Journal: Protein expression and purification

    doi: 10.1016/j.pep.2014.02.015

    PDZ Domain Peptide Ligand Affinity Tags: Fusion of GluN2B Peptide Ligand Truncation Library to GST
    Figure Legend Snippet: PDZ Domain Peptide Ligand Affinity Tags: Fusion of GluN2B Peptide Ligand Truncation Library to GST

    Techniques Used:

    Related Articles

    Affinity Chromatography:

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase
    Article Snippet: .. The MBP-CpPKS1-AL-ACP fusion protein was purified using amylose resin-based affinity chromatography according to the manufacturer’s standard protocol (New England Biolabs). .. SDS-PAGE analysis of the purified protein revealed two distinct bands; one corresponding to full-length MBP-fused CpPKS1-AL-ACP (~138-kDa) and another at approximately 80-kDa.

    Fluorescence:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Mutagenesis:

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice
    Article Snippet: .. CCP6-H230S/E233Q mutant (CCP6mut) and CCP6wt were also subcloned into H-MBP-3C vector and purified using amylose resin (New England BioLabs, Ipswich, USA) according to the manufacturer’s instruction. .. CCP1, TTLL4, Cullin1, and Skp1 were cloned into p3×flag-CMV-9 expression vector. pMX5-Oct4, pMX5-Sox2, pMX5-Klf4, and pMX5-cMyc plasmids were from Dr. Yang Xu’s lab (University of California, San Diego, La Jolla, USA). pDEST-Flag-PHF13-βTrCP1 plasmid was from Dr. Degui Chen’s lab (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China).

    Construct:

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1
    Article Snippet: .. MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer. ..

    Purification:

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice
    Article Snippet: .. CCP6-H230S/E233Q mutant (CCP6mut) and CCP6wt were also subcloned into H-MBP-3C vector and purified using amylose resin (New England BioLabs, Ipswich, USA) according to the manufacturer’s instruction. .. CCP1, TTLL4, Cullin1, and Skp1 were cloned into p3×flag-CMV-9 expression vector. pMX5-Oct4, pMX5-Sox2, pMX5-Klf4, and pMX5-cMyc plasmids were from Dr. Yang Xu’s lab (University of California, San Diego, La Jolla, USA). pDEST-Flag-PHF13-βTrCP1 plasmid was from Dr. Degui Chen’s lab (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China).

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1
    Article Snippet: .. MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer. ..

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase
    Article Snippet: .. The MBP-CpPKS1-AL-ACP fusion protein was purified using amylose resin-based affinity chromatography according to the manufacturer’s standard protocol (New England Biolabs). .. SDS-PAGE analysis of the purified protein revealed two distinct bands; one corresponding to full-length MBP-fused CpPKS1-AL-ACP (~138-kDa) and another at approximately 80-kDa.

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: .. To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation. .. Bacterial-expressed His-MBP-Kindlin-2 bound to MBP Affinity Matrix or His-Select HF Nickel Affinity Gel was incubated with GST-Smurf1 or GST for 12 h at 4°C.

    Article Title: Structure–function insights into direct lipid transfer between membranes by Mmm1–Mdm12 of ERMES
    Article Snippet: .. MBP was expressed in the same manner as other proteins and purified by amylose resin (NEB) in TBS150 containing 1 mM EDTA by using elution buffer (20 mM maltose and 1 mM EDTA in TBS150). ..

    Incubation:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: .. To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation. .. Bacterial-expressed His-MBP-Kindlin-2 bound to MBP Affinity Matrix or His-Select HF Nickel Affinity Gel was incubated with GST-Smurf1 or GST for 12 h at 4°C.

    Activity Assay:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Recombinant:

    Article Title: LncRNA MACC1-AS1 sponges multiple miRNAs and RNA-binding protein PTBP1
    Article Snippet: .. Briefly, recombinant MBP-MCP-conjugated amylose resin (NEB, USA) was prepared at 4 °C. .. Cell lysates prepared from cells expressing MS2-tagged MACC1-AS1 or MACC1-AS1 mutants were incubated with MBP-MCP-coated amylose resin at 4 °C for 4 h in the presence of RNase and protease inhibitors.

    Lysis:

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1
    Article Snippet: .. MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer. ..

    Binding Assay:

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
    Article Snippet: .. Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein. .. The resin was then washed extensively with ACB (sixty-times resin volume), and a small aliquot was viewed under a fluorescence microscope (AxioVert 200M, Zeiss) with excitation at 489 nm and a filter for emission at 520 nm.

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation
    Article Snippet: .. To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation. .. Bacterial-expressed His-MBP-Kindlin-2 bound to MBP Affinity Matrix or His-Select HF Nickel Affinity Gel was incubated with GST-Smurf1 or GST for 12 h at 4°C.

    Plasmid Preparation:

    Article Title: Klf4 glutamylation is required for cell reprogramming and early embryonic development in mice
    Article Snippet: .. CCP6-H230S/E233Q mutant (CCP6mut) and CCP6wt were also subcloned into H-MBP-3C vector and purified using amylose resin (New England BioLabs, Ipswich, USA) according to the manufacturer’s instruction. .. CCP1, TTLL4, Cullin1, and Skp1 were cloned into p3×flag-CMV-9 expression vector. pMX5-Oct4, pMX5-Sox2, pMX5-Klf4, and pMX5-cMyc plasmids were from Dr. Yang Xu’s lab (University of California, San Diego, La Jolla, USA). pDEST-Flag-PHF13-βTrCP1 plasmid was from Dr. Degui Chen’s lab (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs amylose resin based affinity chromatography
    SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach <t>(amylose</t> <t>resin-based</t> <t>affinity</t> <t>chromatography</t> and PAGE gel extraction). The full-length fusion protein (138-kDa) was used in all enzymatic assays. M = protein marker, lane 1 = full length fusion protein from gel purification, lane 2 = amylose resin-based affinity purified protein.
    Amylose Resin Based Affinity Chromatography, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amylose resin based affinity chromatography/product/New England Biolabs
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    amylose resin based affinity chromatography - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach (amylose resin-based affinity chromatography and PAGE gel extraction). The full-length fusion protein (138-kDa) was used in all enzymatic assays. M = protein marker, lane 1 = full length fusion protein from gel purification, lane 2 = amylose resin-based affinity purified protein.

    Journal: International journal for parasitology

    Article Title: Functional Characterization of the Acyl-[Acyl Carrier Protein] Ligase in the Cryptosporidium parvum Giant Polyketide Synthase

    doi: 10.1016/j.ijpara.2006.10.014

    Figure Lengend Snippet: SDS-PAGE analysis of the maltose binding protein-CpPKS1-acyl ligase-acyl carrier protein fusion protein purified by a two-step approach (amylose resin-based affinity chromatography and PAGE gel extraction). The full-length fusion protein (138-kDa) was used in all enzymatic assays. M = protein marker, lane 1 = full length fusion protein from gel purification, lane 2 = amylose resin-based affinity purified protein.

    Article Snippet: The MBP-CpPKS1-AL-ACP fusion protein was purified using amylose resin-based affinity chromatography according to the manufacturer’s standard protocol (New England Biolabs).

    Techniques: SDS Page, Binding Assay, Purification, Affinity Chromatography, Polyacrylamide Gel Electrophoresis, Gel Extraction, Marker, Gel Purification, Affinity Purification

    hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p

    Journal: eLife

    Article Title: An unfolded protein-induced conformational switch activates mammalian IRE1

    doi: 10.7554/eLife.30700

    Figure Lengend Snippet: hIRE1α cLD shows preference for arginines and aromatic residues. ( A ) Comparison of the amino acid preferences of MBP-hIRE1α cLD (blue) and His 10 -BiP (gray) with the amino acid composition of all peptides displayed on the array (total, black). The frequency of each amino acid present in peptides with top 10% binding score is shown for hIRE1α cLD and BiP. The experimental error is calculated from three experimental replicates. Blue stars depict the amino acids that are significantly enriched or depleted in hIRE1α cLD binders (p

    Article Snippet: MBP-IRE1 cLD constructs were purified on an MBP-amylose resin (New England Biolabs) and eluted with 10 mM amylose in Elution Buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 4 mM DTT) after washing the column with 20 column volumes of Lysis Buffer.

    Techniques: Binding Assay

    Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

    Journal: The Journal of Cell Biology

    Article Title: Smurf1 inhibits integrin activation by controlling Kindlin-2 ubiquitination and degradation

    doi: 10.1083/jcb.201609073

    Figure Lengend Snippet: Smurf1 interacts with Kindlin-2 in vivo and in vitro. (A) HEK293T cells were transfected with Flag-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody or normal IgG followed by immunoblotting using Smurf1 antibody. (B) The endogenous interaction between Kindlin-2 and Smurf1 was analyzed by coIP. (C) Fusion protein His-MBP-Kindlin-2 was incubated with GST or GST-Smurf1 in vitro for MBP pull-down assays. Affinity matrices for MBP were used. (D) HEK293T cells were cotransfected with Flag-Smurf2 and GFP-Kindlin-2. 48 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using GFP antibody. (E) Colocalization of endogenous Smurf1 and Kindlin-2 was analyzed by immunofluorescence staining. The image was merged. Bars, 10 µm. (F) Indicated truncates of Smurf1 and Kindlin-2 were constructed according to their functional domains. (G and H) HEK293T cells were transfected with the indicated truncates of Smurf1. Cell lysates were immunoprecipitated with anti-Flag antibody (G) or Kindlin-2 antibody (H) followed by immunoblotting using an anti–Kindlin-2 (G) or Myc (H) antibody. (I) HEK293T cells were transfected with the indicated truncates of GFP-Kindlin-2. Cell lysates were then incubated with GST or GST-Smurf1 in vitro for GST pull-down assays followed by immunoblotting using an anti-GFP antibody. (J) HEK293T cells were transfected with the indicated truncates of Flag-Kindlin-2, and cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting using anti-Myc antibody. (K) The PY motif mutant of Kindlin-2 or Kindlin-2 WT was cotransfected with Smurf1 into HEK293T cells. CoIP was performed with an anti-Flag antibody followed by immunoblotting using an anti-Myc antibody.

    Article Snippet: To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation.

    Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Incubation, Immunofluorescence, Staining, Construct, Functional Assay, Mutagenesis

    On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.

    Journal: PLoS ONE

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    doi: 10.1371/journal.pone.0008381

    Figure Lengend Snippet: On-column production of M L protein. A. The co-purification labeling process was monitored through SDS-PAGE analysis with Coomassie blue staining or fluorescence scanning as indicated. Lanes 1 and 2: total E. coli proteins before and after IPTG-induced expression of the precursor protein MI N C, respectively. Lane 3: soluble fraction of the cell lysate of lane 2. Lane 4: proteins of lane 3 bound to chitin beads. Lane 5: proteins released from the chitin beads of lane 4 after an overnight incubation with the labeling peptide I C -L in the presence of TCEP. B. Amylose resin was incubated with (panels 1 and 2) or without (panels 3 and 4) the purified M L protein. Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence images. C. SDS-PAGE analysis of the M L protein fractions eluted from the amylose resin of panels 3 and 4 of B using a maltose-containing elution buffer.

    Article Snippet: Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein.

    Techniques: Copurification, Labeling, SDS Page, Staining, Fluorescence, Expressing, Incubation, Purification

    Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.

    Journal: PLoS ONE

    Article Title: Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    doi: 10.1371/journal.pone.0008381

    Figure Lengend Snippet: Protein C-terminal labeling with a fluorophore. A . Schematic illustration of the labeling reaction. I N and I C : components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide I C -L, I C is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B . Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MI N C), the labeled protein (M L ), and the excised N-intein (I N C) are indicated. In lane 1, a minor protein band at the same position as M L is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MI N C precursor protein and I C -L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.

    Article Snippet: Activity of the fluorescence-labeled maltose binding protein (ML ) Amylose resin (New England Biolabs) was equilibrated with Amylose Column Buffer (ACB: 20 mM Tris-HCl, 200 mM NaCl; pH 7.4) and incubated for 1 h at 4°C with ML protein.

    Techniques: Labeling, Binding Assay, Purification, Sequencing, Incubation, SDS Page, Staining, Western Blot, Fluorescence, Affinity Chromatography