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rabbit anti sumo1  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc rabbit anti sumo1
    Rabbit Anti Sumo1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti sumo1/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    rabbit anti sumo1 - by Bioz Stars, 2025-02
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    Screening of new covalent inhibitors targeting the allosteric pocket of SUMO E1. (a) A schematic diagram of the SUMOylation pathway. SUMO E1 is a heterodimer of Aos1 and Uba2. Ubc9 is the sole SUMO E2 discovered thus far. (b) The complex of SUMO E1 and a covalent allosteric inhibitor (CAI) COH000 (PDB ID: 6CWY). COH000 is shown in sticks and formed a covalent bond with Uba2-C30. The residues of the allosteric pocket are shown in surface, and the residues of the ATP-binding pocket are shown in dotted surface. The SUMO-binding site is far away from the allosteric pocket and not shown. (c) MCULE-3064932370 and MCULE-6830015021 inhibited the formation of the SUMOylation of Uba2. Before the addition of <t>SUMO1,</t> the compounds were pre-incubated with SUMO E1 (Aos1/Uba2) at 37 °C for 30 minutes at various concentrations according to their solubility. MCULE-3064932370 and MCULE-6830015021 were 100 µM, MCULE-6321090433 was 50 µM, and the others were 500 µM. The reductant DTT (dithiothreitol) inhibits SUMOylation and was used as a control. The results with 60 min and 90 min pre-incubation had similar results (Figure S1, S2). All samples had 1% (v/v) DMSO. (d) Time-dependent and concentration-dependent inhibition of MCULE-3064932370 and MCULE-6830015021 on the SUMOylation of Uba2. The quantitative data from three replicates are shown in (e). Error bars represent mean ± s.d.. (f) Representative spectra from the full-protein mass spectrometry analysis. (g) Statistical analysis of the m/z data from multiple MALDI-TOF detections (n ≥ 6).
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    Screening of new covalent inhibitors targeting the allosteric pocket of SUMO E1. (a) A schematic diagram of the SUMOylation pathway. SUMO E1 is a heterodimer of Aos1 and Uba2. Ubc9 is the sole SUMO E2 discovered thus far. (b) The complex of SUMO E1 and a covalent allosteric inhibitor (CAI) COH000 (PDB ID: 6CWY). COH000 is shown in sticks and formed a covalent bond with Uba2-C30. The residues of the allosteric pocket are shown in surface, and the residues of the ATP-binding pocket are shown in dotted surface. The SUMO-binding site is far away from the allosteric pocket and not shown. (c) MCULE-3064932370 and MCULE-6830015021 inhibited the formation of the SUMOylation of Uba2. Before the addition of <t>SUMO1,</t> the compounds were pre-incubated with SUMO E1 (Aos1/Uba2) at 37 °C for 30 minutes at various concentrations according to their solubility. MCULE-3064932370 and MCULE-6830015021 were 100 µM, MCULE-6321090433 was 50 µM, and the others were 500 µM. The reductant DTT (dithiothreitol) inhibits SUMOylation and was used as a control. The results with 60 min and 90 min pre-incubation had similar results (Figure S1, S2). All samples had 1% (v/v) DMSO. (d) Time-dependent and concentration-dependent inhibition of MCULE-3064932370 and MCULE-6830015021 on the SUMOylation of Uba2. The quantitative data from three replicates are shown in (e). Error bars represent mean ± s.d.. (f) Representative spectra from the full-protein mass spectrometry analysis. (g) Statistical analysis of the m/z data from multiple MALDI-TOF detections (n ≥ 6).
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    Screening of new covalent inhibitors targeting the allosteric pocket of SUMO E1. (a) A schematic diagram of the SUMOylation pathway. SUMO E1 is a heterodimer of Aos1 and Uba2. Ubc9 is the sole SUMO E2 discovered thus far. (b) The complex of SUMO E1 and a covalent allosteric inhibitor (CAI) COH000 (PDB ID: 6CWY). COH000 is shown in sticks and formed a covalent bond with Uba2-C30. The residues of the allosteric pocket are shown in surface, and the residues of the ATP-binding pocket are shown in dotted surface. The SUMO-binding site is far away from the allosteric pocket and not shown. (c) MCULE-3064932370 and MCULE-6830015021 inhibited the formation of the SUMOylation of Uba2. Before the addition of <t>SUMO1,</t> the compounds were pre-incubated with SUMO E1 (Aos1/Uba2) at 37 °C for 30 minutes at various concentrations according to their solubility. MCULE-3064932370 and MCULE-6830015021 were 100 µM, MCULE-6321090433 was 50 µM, and the others were 500 µM. The reductant DTT (dithiothreitol) inhibits SUMOylation and was used as a control. The results with 60 min and 90 min pre-incubation had similar results (Figure S1, S2). All samples had 1% (v/v) DMSO. (d) Time-dependent and concentration-dependent inhibition of MCULE-3064932370 and MCULE-6830015021 on the SUMOylation of Uba2. The quantitative data from three replicates are shown in (e). Error bars represent mean ± s.d.. (f) Representative spectra from the full-protein mass spectrometry analysis. (g) Statistical analysis of the m/z data from multiple MALDI-TOF detections (n ≥ 6).
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    a Ni 2+ -NTA bead affinity pull-down assay results showing the modification of exogenous huANP32A (left) and huANP32B (right) with <t>SUMO1,</t> SUMO2, and SUMO3 in HEK293T cells. b Ni 2+ -NTA bead affinity pull-down assay results showing the modification of endogenous huANP32A and huANP32B with SUMO1 in HEK293T cells. *indicates non-specific protein band. c Western blot analysis showing that H9N2 virus infection upregulates the global SUMOylation level in HEK293T cells. d Ni 2+ -NTA bead affinity pull-down assay results showing that the SUMOylation of huANP32A (left) or huANP32B (right) in HEK293T cells was notably enhanced by H9N2 AIV infection. HEK293T cells were transfected with the indicated plasmids for 24 hours followed by H9N2 AIV infection (MOI = 0.01) for another 24 hours. Cells were then harvested for SUMOylation assays and immunoblotting analysis. In ( a to d ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.
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    a Ni 2+ -NTA bead affinity pull-down assay results showing the modification of exogenous huANP32A (left) and huANP32B (right) with <t>SUMO1,</t> SUMO2, and SUMO3 in HEK293T cells. b Ni 2+ -NTA bead affinity pull-down assay results showing the modification of endogenous huANP32A and huANP32B with SUMO1 in HEK293T cells. *indicates non-specific protein band. c Western blot analysis showing that H9N2 virus infection upregulates the global SUMOylation level in HEK293T cells. d Ni 2+ -NTA bead affinity pull-down assay results showing that the SUMOylation of huANP32A (left) or huANP32B (right) in HEK293T cells was notably enhanced by H9N2 AIV infection. HEK293T cells were transfected with the indicated plasmids for 24 hours followed by H9N2 AIV infection (MOI = 0.01) for another 24 hours. Cells were then harvested for SUMOylation assays and immunoblotting analysis. In ( a to d ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.
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    SUMOylation inhibitor, ML-792, reverses the 5-FU resistance in HCT-8/5-FU cell line. (A) SUMOylation inhibitor ML-792 inhibits the conjunction of <t>SUMO1</t> and SUMO2/3 to substrates. (B) 5-FU IC 50 detection was conducted in HCT-8 cells with incubation of 0.1 µM ML-792 or DMSO for control. (C) 1,000 cells/well of HCT-8/5-FU were seeded in 6-well plate for colony formation assay with combination incubation of 100 µM 5-FU and 0, one or 5 µM ML-792. * p < 0.05, ** p < 0.01.
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    SUMOylation inhibitor, ML-792, reverses the 5-FU resistance in HCT-8/5-FU cell line. (A) SUMOylation inhibitor ML-792 inhibits the conjunction of <t>SUMO1</t> and SUMO2/3 to substrates. (B) 5-FU IC 50 detection was conducted in HCT-8 cells with incubation of 0.1 µM ML-792 or DMSO for control. (C) 1,000 cells/well of HCT-8/5-FU were seeded in 6-well plate for colony formation assay with combination incubation of 100 µM 5-FU and 0, one or 5 µM ML-792. * p < 0.05, ** p < 0.01.
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    Cell Signaling Technology Inc sumo 1 c9h1 rabbit mab
    SUMOylation inhibitor, ML-792, reverses the 5-FU resistance in HCT-8/5-FU cell line. (A) SUMOylation inhibitor ML-792 inhibits the conjunction of <t>SUMO1</t> and SUMO2/3 to substrates. (B) 5-FU IC 50 detection was conducted in HCT-8 cells with incubation of 0.1 µM ML-792 or DMSO for control. (C) 1,000 cells/well of HCT-8/5-FU were seeded in 6-well plate for colony formation assay with combination incubation of 100 µM 5-FU and 0, one or 5 µM ML-792. * p < 0.05, ** p < 0.01.
    Sumo 1 C9h1 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Screening of new covalent inhibitors targeting the allosteric pocket of SUMO E1. (a) A schematic diagram of the SUMOylation pathway. SUMO E1 is a heterodimer of Aos1 and Uba2. Ubc9 is the sole SUMO E2 discovered thus far. (b) The complex of SUMO E1 and a covalent allosteric inhibitor (CAI) COH000 (PDB ID: 6CWY). COH000 is shown in sticks and formed a covalent bond with Uba2-C30. The residues of the allosteric pocket are shown in surface, and the residues of the ATP-binding pocket are shown in dotted surface. The SUMO-binding site is far away from the allosteric pocket and not shown. (c) MCULE-3064932370 and MCULE-6830015021 inhibited the formation of the SUMOylation of Uba2. Before the addition of SUMO1, the compounds were pre-incubated with SUMO E1 (Aos1/Uba2) at 37 °C for 30 minutes at various concentrations according to their solubility. MCULE-3064932370 and MCULE-6830015021 were 100 µM, MCULE-6321090433 was 50 µM, and the others were 500 µM. The reductant DTT (dithiothreitol) inhibits SUMOylation and was used as a control. The results with 60 min and 90 min pre-incubation had similar results (Figure S1, S2). All samples had 1% (v/v) DMSO. (d) Time-dependent and concentration-dependent inhibition of MCULE-3064932370 and MCULE-6830015021 on the SUMOylation of Uba2. The quantitative data from three replicates are shown in (e). Error bars represent mean ± s.d.. (f) Representative spectra from the full-protein mass spectrometry analysis. (g) Statistical analysis of the m/z data from multiple MALDI-TOF detections (n ≥ 6).

    Journal: bioRxiv

    Article Title: SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis

    doi: 10.1101/2024.12.12.627095

    Figure Lengend Snippet: Screening of new covalent inhibitors targeting the allosteric pocket of SUMO E1. (a) A schematic diagram of the SUMOylation pathway. SUMO E1 is a heterodimer of Aos1 and Uba2. Ubc9 is the sole SUMO E2 discovered thus far. (b) The complex of SUMO E1 and a covalent allosteric inhibitor (CAI) COH000 (PDB ID: 6CWY). COH000 is shown in sticks and formed a covalent bond with Uba2-C30. The residues of the allosteric pocket are shown in surface, and the residues of the ATP-binding pocket are shown in dotted surface. The SUMO-binding site is far away from the allosteric pocket and not shown. (c) MCULE-3064932370 and MCULE-6830015021 inhibited the formation of the SUMOylation of Uba2. Before the addition of SUMO1, the compounds were pre-incubated with SUMO E1 (Aos1/Uba2) at 37 °C for 30 minutes at various concentrations according to their solubility. MCULE-3064932370 and MCULE-6830015021 were 100 µM, MCULE-6321090433 was 50 µM, and the others were 500 µM. The reductant DTT (dithiothreitol) inhibits SUMOylation and was used as a control. The results with 60 min and 90 min pre-incubation had similar results (Figure S1, S2). All samples had 1% (v/v) DMSO. (d) Time-dependent and concentration-dependent inhibition of MCULE-3064932370 and MCULE-6830015021 on the SUMOylation of Uba2. The quantitative data from three replicates are shown in (e). Error bars represent mean ± s.d.. (f) Representative spectra from the full-protein mass spectrometry analysis. (g) Statistical analysis of the m/z data from multiple MALDI-TOF detections (n ≥ 6).

    Article Snippet: The rabbit monoclonal anti-SUMO1 (Cat. NO. HY-P80351) was from MedChemExpress LLC (U.S.A.).

    Techniques: Binding Assay, Incubation, Solubility, Control, Concentration Assay, Inhibition, Mass Spectrometry

    The binding of the new SUMO E1 CAIs depends on the Aos1/Uba2 complex. (a) Representative spectra and statistical analysis data from multiple MALDI-TOF detections (n ≥ 6) of the full-protein mass spectrometry analysis of the Uba2 protein when it was incubated with the inhibitors alone. (b) Representative spectra and statistical analysis data from multiple MALDI-TOF detections (n ≥ 6) of the full-protein mass spectrometry analysis of the Aos1 protein when it was incubated with the inhibitors alone. (c) The thermal denaturation result from the gel-based thermal shift assay of the Aos1/Uba2 complex with the new CAIs. (d) The competition experiment of the CAIs with the substrate SUMO1. (e) The 2D interaction diagrams of Aos1/Uba2 with COH000 (PDB ID: 6CWY), MCULE-3064932370, and MCULE-6830015021. Aos1 is the chain C, and Uba2 is the chain D. Since SCARdock only considers non-covalent interaction, the Uba2-C30 was computationally mutated to G30 as shown in the diagrams. Therefore, the ligands are shown not covalently bonded to Uba2. The ligands and residues are shown in ball-and-stick representation. The green dotted line indicates a hydrogen bond. The spoked arcs represent residues making nonbonded contacts with the ligands. The red circles indicate residues in equivalent 3D positions when the structural models are superposed.

    Journal: bioRxiv

    Article Title: SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis

    doi: 10.1101/2024.12.12.627095

    Figure Lengend Snippet: The binding of the new SUMO E1 CAIs depends on the Aos1/Uba2 complex. (a) Representative spectra and statistical analysis data from multiple MALDI-TOF detections (n ≥ 6) of the full-protein mass spectrometry analysis of the Uba2 protein when it was incubated with the inhibitors alone. (b) Representative spectra and statistical analysis data from multiple MALDI-TOF detections (n ≥ 6) of the full-protein mass spectrometry analysis of the Aos1 protein when it was incubated with the inhibitors alone. (c) The thermal denaturation result from the gel-based thermal shift assay of the Aos1/Uba2 complex with the new CAIs. (d) The competition experiment of the CAIs with the substrate SUMO1. (e) The 2D interaction diagrams of Aos1/Uba2 with COH000 (PDB ID: 6CWY), MCULE-3064932370, and MCULE-6830015021. Aos1 is the chain C, and Uba2 is the chain D. Since SCARdock only considers non-covalent interaction, the Uba2-C30 was computationally mutated to G30 as shown in the diagrams. Therefore, the ligands are shown not covalently bonded to Uba2. The ligands and residues are shown in ball-and-stick representation. The green dotted line indicates a hydrogen bond. The spoked arcs represent residues making nonbonded contacts with the ligands. The red circles indicate residues in equivalent 3D positions when the structural models are superposed.

    Article Snippet: The rabbit monoclonal anti-SUMO1 (Cat. NO. HY-P80351) was from MedChemExpress LLC (U.S.A.).

    Techniques: Binding Assay, Mass Spectrometry, Incubation, Thermal Shift Assay

    The new SUMO E1 CAIs perturb the SUMOylation pathway. T47D cells were treated with MCULE-3064932370 and MCULE-6830015021 for 48 h before evaluation. (a) The cellular levels of RanGAP1, RanGAP1-SUMO, and SUMO1 were assessed by Western blotting. (b) The cellular levels of Uba2 and Ubc9 were assessed by Western blotting.

    Journal: bioRxiv

    Article Title: SUMO E1 covalent allosteric inhibitors modulate polyamine synthesis via the MAT2A-AdoMetDC axis

    doi: 10.1101/2024.12.12.627095

    Figure Lengend Snippet: The new SUMO E1 CAIs perturb the SUMOylation pathway. T47D cells were treated with MCULE-3064932370 and MCULE-6830015021 for 48 h before evaluation. (a) The cellular levels of RanGAP1, RanGAP1-SUMO, and SUMO1 were assessed by Western blotting. (b) The cellular levels of Uba2 and Ubc9 were assessed by Western blotting.

    Article Snippet: The rabbit monoclonal anti-SUMO1 (Cat. NO. HY-P80351) was from MedChemExpress LLC (U.S.A.).

    Techniques: Western Blot

    a Ni 2+ -NTA bead affinity pull-down assay results showing the modification of exogenous huANP32A (left) and huANP32B (right) with SUMO1, SUMO2, and SUMO3 in HEK293T cells. b Ni 2+ -NTA bead affinity pull-down assay results showing the modification of endogenous huANP32A and huANP32B with SUMO1 in HEK293T cells. *indicates non-specific protein band. c Western blot analysis showing that H9N2 virus infection upregulates the global SUMOylation level in HEK293T cells. d Ni 2+ -NTA bead affinity pull-down assay results showing that the SUMOylation of huANP32A (left) or huANP32B (right) in HEK293T cells was notably enhanced by H9N2 AIV infection. HEK293T cells were transfected with the indicated plasmids for 24 hours followed by H9N2 AIV infection (MOI = 0.01) for another 24 hours. Cells were then harvested for SUMOylation assays and immunoblotting analysis. In ( a to d ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

    doi: 10.1038/s41467-024-55034-y

    Figure Lengend Snippet: a Ni 2+ -NTA bead affinity pull-down assay results showing the modification of exogenous huANP32A (left) and huANP32B (right) with SUMO1, SUMO2, and SUMO3 in HEK293T cells. b Ni 2+ -NTA bead affinity pull-down assay results showing the modification of endogenous huANP32A and huANP32B with SUMO1 in HEK293T cells. *indicates non-specific protein band. c Western blot analysis showing that H9N2 virus infection upregulates the global SUMOylation level in HEK293T cells. d Ni 2+ -NTA bead affinity pull-down assay results showing that the SUMOylation of huANP32A (left) or huANP32B (right) in HEK293T cells was notably enhanced by H9N2 AIV infection. HEK293T cells were transfected with the indicated plasmids for 24 hours followed by H9N2 AIV infection (MOI = 0.01) for another 24 hours. Cells were then harvested for SUMOylation assays and immunoblotting analysis. In ( a to d ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

    Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

    Techniques: Pull Down Assay, Modification, Western Blot, Virus, Infection, Transfection

    a Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated constructs on H9N2 vPol activity. Values above K0 were statistically analyzed using one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32A-K0 (error bars represent the mean ± SD of n = 4 independent biological replicates). b Schematic representation of the huANP32A-K0 mutants generated. Western blots demonstrate comparable expression levels for all indicated constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of MDCK-TKO cells stably reconstituted with the indicated huANP32A-K0-Flag constructs or empty vector. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K153 sites of huANP32A can be modified by SUMO1. h Co-IP experiments showing that the K68R/K153R mutations in huANP32A suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32A. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-Flag or huANP32A-K68R/K153R-Flag (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32A-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). In ( g , h ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

    doi: 10.1038/s41467-024-55034-y

    Figure Lengend Snippet: a Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated constructs on H9N2 vPol activity. Values above K0 were statistically analyzed using one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32A-K0 (error bars represent the mean ± SD of n = 4 independent biological replicates). b Schematic representation of the huANP32A-K0 mutants generated. Western blots demonstrate comparable expression levels for all indicated constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of MDCK-TKO cells stably reconstituted with the indicated huANP32A-K0-Flag constructs or empty vector. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K153 sites of huANP32A can be modified by SUMO1. h Co-IP experiments showing that the K68R/K153R mutations in huANP32A suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32A constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32A. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32A-Flag or huANP32A-K68R/K153R-Flag (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32A-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). In ( g , h ), experiments were independently repeated three times with consistent results. Source data are provided as a Source Data file.

    Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

    Techniques: Construct, Activity Assay, Generated, Western Blot, Expressing, Virus, Control, Stable Transfection, Mutagenesis, Plasmid Preparation, Pull Down Assay, Modification, Co-Immunoprecipitation Assay, Two Tailed Test

    a Minigenome assays in HEK293T-TKO cells comparing the effect of indicated huANP32B-K0 constructs on the H9N2 vPol activity. Values higher than K0 were analyzed via one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32B-K0 (error bars represent the mean ± SD of n = 3 independent biological replicates). b Schematic representation of the huANP32B-K0 mutants generated. Western blots confirmed comparable expression levels of all constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-K0-Flag constructs. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K116 sites of huANP32B can be modified by SUMO1. h Co-IP experiments showing that the K68R/K116R mutations in huANP32B suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32B. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). Experiments in ( g and h ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Human ANP32A/B are SUMOylated and utilized by avian influenza virus NS2 protein to overcome species-specific restriction

    doi: 10.1038/s41467-024-55034-y

    Figure Lengend Snippet: a Minigenome assays in HEK293T-TKO cells comparing the effect of indicated huANP32B-K0 constructs on the H9N2 vPol activity. Values higher than K0 were analyzed via one-way ANOVA followed by a Dunnett’s multiple comparisons test against huANP32B-K0 (error bars represent the mean ± SD of n = 3 independent biological replicates). b Schematic representation of the huANP32B-K0 mutants generated. Western blots confirmed comparable expression levels of all constructs. c , d Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B-K0 constructs on the H9N2 ( c ) and H7N9 (PB2-627E) ( d ) vPol activity in the presence of NS2. e The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-K0-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. f Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-K0-Flag constructs. g Ni 2+ -NTA bead affinity pull-down assay showing that the K68 and K116 sites of huANP32B can be modified by SUMO1. h Co-IP experiments showing that the K68R/K116R mutations in huANP32B suppress its interaction with H9N2-NS2. i , j Minigenome assays in HEK293T-TKO cells comparing the effect of the indicated huANP32B constructs on the H9N2 ( i ) and H7N9 (PB2-627E) ( j ) vPol activity. Statistical analyses were performed relative to huANP32B. k The replication kinetics of the avian H9N2 virus were evaluated in control MDCK-TKO cells or those stably expressing huANP32B-Flag or its mutant (MOI = 0.01), with viral titers determined at the indicated time points. l Western blot analysis of control MDCK-TKO cells or those stably expressing indicated huANP32B-Flag constructs. In ( c to e ) and ( i to k ), error bars represent the mean ± SD of n = 3 independent biological replicates; NS, not significant; Statistical significance was determined by two-tailed unpaired t-test ( c , d , i and j ) or two-way ANOVA ( k ). Experiments in ( g and h ) were independently repeated three times with consistent results. Source data are provided as a Source Data file.

    Article Snippet: Western blot analysis was conducted following established protocols using the following antibodies: rabbit anti-Flag (Sigma, F7425), mouse anti-Flag (Sigma, F1804), rabbit anti-HA (Sigma, H6908), rabbit anti-ACTB (Abclonal, AC026), mouse anti-ACTB (Abclonal, AC004), rabbit anti-Myc (Abclonal, AE070), rabbit anti-SUMO1 (Abclonal, A19121), rabbit anti-SUMO2/3 (Abclonal, A5066), rabbit anti-SENP1(Abcam, ab108981), mouse anti-His (Proteintech, 66005-1-Ig), rabbit anti-V5 (Proteintech, 14440-1-AP), rabbit anti-SENP1 (Proteintech, 25349-1-AP), rabbit anti-PIAS2 (Proteintech, 16074-1-AP), rabbit anti-GST (Proteintech, 10000-0-AP), rabbit anti-ANP32A (Proteintech,15810-1-AP), rabbit anti-ANP32B (Proteintech, 10843-1-AP), mouse anti-ANP32A (Proteintech, 67687-1-Ig), mouse anti-ANP32B (Proteintech, 66160-1-Ig), rabbit anti-influenza A virus NS2 (GeneTex, GTX125953), rabbit anti-influenza B virus NP (GeneTex, GTX128538), rabbit anti-influenza A virus PB2 (GeneTex, GTX125926), rabbit anti-influenza A virus PA (GeneTex, GTX118991), mouse anti-influenza A virus PA (prepared in our laboratory, 1:5000 for WB), mouse anti-influenza A virus NP (prepared in our laboratory, 1:5000 for WB), Biotin-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00004-2), DyLight 800-labeled Anti-Mouse IgG (H + L) Antibody (KPL, 5230-0415), DyLight 680-labeled Anti-Rabbit IgG (H + L) Antibody (KPL, 5230-0402) and DyLight™ 680-labeled streptavidin (KPL, 5270-0025).

    Techniques: Construct, Activity Assay, Generated, Western Blot, Expressing, Virus, Control, Stable Transfection, Mutagenesis, Pull Down Assay, Modification, Co-Immunoprecipitation Assay, Two Tailed Test

    SUMOylation inhibitor, ML-792, reverses the 5-FU resistance in HCT-8/5-FU cell line. (A) SUMOylation inhibitor ML-792 inhibits the conjunction of SUMO1 and SUMO2/3 to substrates. (B) 5-FU IC 50 detection was conducted in HCT-8 cells with incubation of 0.1 µM ML-792 or DMSO for control. (C) 1,000 cells/well of HCT-8/5-FU were seeded in 6-well plate for colony formation assay with combination incubation of 100 µM 5-FU and 0, one or 5 µM ML-792. * p < 0.05, ** p < 0.01.

    Journal: Frontiers in Pharmacology

    Article Title: RREB1-mediated SUMOylation enhancement promotes chemoresistance partially by transcriptionally upregulating UBC9 in colorectal cancer

    doi: 10.3389/fphar.2024.1381860

    Figure Lengend Snippet: SUMOylation inhibitor, ML-792, reverses the 5-FU resistance in HCT-8/5-FU cell line. (A) SUMOylation inhibitor ML-792 inhibits the conjunction of SUMO1 and SUMO2/3 to substrates. (B) 5-FU IC 50 detection was conducted in HCT-8 cells with incubation of 0.1 µM ML-792 or DMSO for control. (C) 1,000 cells/well of HCT-8/5-FU were seeded in 6-well plate for colony formation assay with combination incubation of 100 µM 5-FU and 0, one or 5 µM ML-792. * p < 0.05, ** p < 0.01.

    Article Snippet: Specific primary antibodies: rabbit anti-SUMO1 (ET1606-53, HuaBio), rabbit anti-SUMO2/3 (ET1701-17, HuaBio), rabbit anti-UBC9 (ET1610-21, HuaBio), rabbit anti-SAE1 (ET7108-22, HuaBio), rabbit anti-SAE2 (ET1705-73, HuaBio), mouse anti-Flag (F1804, Sigma-Aldrich).

    Techniques: Incubation, Control, Colony Assay

    Overexpression of UBC9 or SAE1 enhances the 5-FU resistance in HCT116 cells. (A) Overexpression of HA-UBC9 or Flag-SAE1 in HCT116 cells increased more SUMO2/3 modified protein levels than that of SUMO1. (B,C) 5-FU IC 50 detection in HCT-8 cells with UBC9 overexpression or SAE1 overexpression. Y -axis was shown as percentage of control group (without 5-FU treatment). (D) Colony formation assay to examine the survival of HCT116 cells under 0.5 µM 5-FU treatment with or without overexpression of UBC9 or SAE1. (E) The clone survival numbers in colony formation assay performed in Figure (D) * p < 0.05.

    Journal: Frontiers in Pharmacology

    Article Title: RREB1-mediated SUMOylation enhancement promotes chemoresistance partially by transcriptionally upregulating UBC9 in colorectal cancer

    doi: 10.3389/fphar.2024.1381860

    Figure Lengend Snippet: Overexpression of UBC9 or SAE1 enhances the 5-FU resistance in HCT116 cells. (A) Overexpression of HA-UBC9 or Flag-SAE1 in HCT116 cells increased more SUMO2/3 modified protein levels than that of SUMO1. (B,C) 5-FU IC 50 detection in HCT-8 cells with UBC9 overexpression or SAE1 overexpression. Y -axis was shown as percentage of control group (without 5-FU treatment). (D) Colony formation assay to examine the survival of HCT116 cells under 0.5 µM 5-FU treatment with or without overexpression of UBC9 or SAE1. (E) The clone survival numbers in colony formation assay performed in Figure (D) * p < 0.05.

    Article Snippet: Specific primary antibodies: rabbit anti-SUMO1 (ET1606-53, HuaBio), rabbit anti-SUMO2/3 (ET1701-17, HuaBio), rabbit anti-UBC9 (ET1610-21, HuaBio), rabbit anti-SAE1 (ET7108-22, HuaBio), rabbit anti-SAE2 (ET1705-73, HuaBio), mouse anti-Flag (F1804, Sigma-Aldrich).

    Techniques: Over Expression, Modification, Control, Colony Assay