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Cell Signaling Technology Inc rabbit polyclonal abs
Rabbit Polyclonal Abs, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal abs/product/Cell Signaling Technology Inc
Average 91 stars, based on 10 article reviews
Price from $9.99 to $1999.99
rabbit polyclonal abs - by Bioz Stars, 2020-09
91/100 stars

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Related Articles

Immunohistochemistry:

Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1
Article Snippet: .. Slides were then washed and incubated with M.O.M. biotinylated anti-mouse IgG reagent (1/250 dilution; Vector Laboratories) for 10 min. For IHC for caspase-8 or caspase-3, sections were incubated overnight with either a rabbit polyclonal Ab for cleaved caspase-3 (1/200 dilution; Cell Signaling Technologies) (data not shown) or a rabbit polyclonal Ab for active/cleaved caspase-8 (1/200 dilution; Imgenex) followed by incubation with a biotinylated goat anti-rabbit IgG (1/500; Santa Cruz Biotechnology) for 1 h. Immunoreactivity was demonstrated using the ABC immunostaining system kit (ABC kit; Vector Laboratories) for Fas, caspase-8, and caspase-3 or using the avidin biotin complex immunoperoxidase system from the M.O.M. immunodetection kit PK-2200 (Vector Laboratories) for FasL analysis. .. Color development was completed with the 3′,3-diaminobenzidine substrate (Sigma-Aldrich), and sections were counterstained with hematoxylin.

Immunodetection:

Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1
Article Snippet: .. Slides were then washed and incubated with M.O.M. biotinylated anti-mouse IgG reagent (1/250 dilution; Vector Laboratories) for 10 min. For IHC for caspase-8 or caspase-3, sections were incubated overnight with either a rabbit polyclonal Ab for cleaved caspase-3 (1/200 dilution; Cell Signaling Technologies) (data not shown) or a rabbit polyclonal Ab for active/cleaved caspase-8 (1/200 dilution; Imgenex) followed by incubation with a biotinylated goat anti-rabbit IgG (1/500; Santa Cruz Biotechnology) for 1 h. Immunoreactivity was demonstrated using the ABC immunostaining system kit (ABC kit; Vector Laboratories) for Fas, caspase-8, and caspase-3 or using the avidin biotin complex immunoperoxidase system from the M.O.M. immunodetection kit PK-2200 (Vector Laboratories) for FasL analysis. .. Color development was completed with the 3′,3-diaminobenzidine substrate (Sigma-Aldrich), and sections were counterstained with hematoxylin.

Immunostaining:

Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1
Article Snippet: .. Slides were then washed and incubated with M.O.M. biotinylated anti-mouse IgG reagent (1/250 dilution; Vector Laboratories) for 10 min. For IHC for caspase-8 or caspase-3, sections were incubated overnight with either a rabbit polyclonal Ab for cleaved caspase-3 (1/200 dilution; Cell Signaling Technologies) (data not shown) or a rabbit polyclonal Ab for active/cleaved caspase-8 (1/200 dilution; Imgenex) followed by incubation with a biotinylated goat anti-rabbit IgG (1/500; Santa Cruz Biotechnology) for 1 h. Immunoreactivity was demonstrated using the ABC immunostaining system kit (ABC kit; Vector Laboratories) for Fas, caspase-8, and caspase-3 or using the avidin biotin complex immunoperoxidase system from the M.O.M. immunodetection kit PK-2200 (Vector Laboratories) for FasL analysis. .. Color development was completed with the 3′,3-diaminobenzidine substrate (Sigma-Aldrich), and sections were counterstained with hematoxylin.

Avidin-Biotin Assay:

Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1
Article Snippet: .. Slides were then washed and incubated with M.O.M. biotinylated anti-mouse IgG reagent (1/250 dilution; Vector Laboratories) for 10 min. For IHC for caspase-8 or caspase-3, sections were incubated overnight with either a rabbit polyclonal Ab for cleaved caspase-3 (1/200 dilution; Cell Signaling Technologies) (data not shown) or a rabbit polyclonal Ab for active/cleaved caspase-8 (1/200 dilution; Imgenex) followed by incubation with a biotinylated goat anti-rabbit IgG (1/500; Santa Cruz Biotechnology) for 1 h. Immunoreactivity was demonstrated using the ABC immunostaining system kit (ABC kit; Vector Laboratories) for Fas, caspase-8, and caspase-3 or using the avidin biotin complex immunoperoxidase system from the M.O.M. immunodetection kit PK-2200 (Vector Laboratories) for FasL analysis. .. Color development was completed with the 3′,3-diaminobenzidine substrate (Sigma-Aldrich), and sections were counterstained with hematoxylin.

FLAG-tag:

Article Title: STIM2 interacts with AMPK and regulates calcium-induced AMPK activation
Article Snippet: .. The following antibodies were used in our study: phospho–AMPK-α (2535S, 1:1000 dilution; Cell Signaling Technology) rabbit mAb, AMPK-α (5832S, 1:1000 dilution; Cell Signaling Technology) rabbit mAb, AMPK-β (4150S, 1:1000 dilution; Cell Signaling Technology) rabbit mAb, AMPK-γ (4187S, 1:1000 dilution; Cell Signaling Technology) rabbit polyclonal Ab, phospho-ACC (3661S, 1:1000 dilution; Cell Signaling Technology) rabbit pAb, acetylCoA carboxylase (ACC) (3662S, 1:1000 dilution; Cell Signaling Technology) rabbit pAb, phospho-S6K (9205S, 1:1000 dilution; Cell Signaling Technology) rabbit pAb, S6K (9202S, 1:1000 dilution; Cell Signaling Technology) rabbit pAb, phospho-S6 (2215S, 1:3000 dilution; Cell Signaling Technology) rabbit pAb, S6 (2217S, 1:5000 dilution; Cell Signaling Technology) rabbit mAb, STIM1 (5668S, 1:1000 dilution; Cell Signaling Technology) rabbit mAb, STIM2 (ACC-064, 1:1000 dilution; Alomone Labs, Jerusalem, Israel) rabbit polyclonal Ab, vinculin (V4505, 1:10,000 dilution; MilliporeSigma) mouse mAb, c-Myc (5605P, 1:1000 dilution; Cell Signaling Technology) rabbit mAb, and Flag-tag (14793S, 1:5000 dilution; Cell Signaling Technology) rabbit mAb. .. Cell death assay Cell death was measured by propidium iodide (PI) staining as previously described ( , , ).

Incubation:

Article Title: The neuro-steroid, 3β androstene 17α diol exhibits potent cytotoxic effects on human malignant glioma and lymphoma cells through different programmed cell death pathways
Article Snippet: .. Membranes were blocked with 5% non-fat dry milk+0.1% Tween-20 in PBS for 1 h. The membranes were incubated overnight with rabbit polyclonal Ab specific for poly (ADP-ribose) polymerase (PARP) or caspase 3 (both from Cell Signaling Technology, Beverly, MA, USA) at 4°C, washed with 0.1% Tween-20 in PBS and incubated with goat anti-rabbit Ab conjugated to horseradish peroxidase (Rockland Immunochemicals, Gilbertsville, PA, USA) for 1 h. Immunoreactive bands were visualized by chemiluminescence (SuperSignal, Pierce Chemicals). .. The same membranes were reblotted with a mouse anti-β -actin mAb (Sigma, St Louis, MO, USA), as described above, and used as a loading control.

Article Title: Differential Role of the Fas/Fas Ligand Apoptotic Pathway in Inflammation and Lung Fibrosis Associated with Reovirus 1/L-Induced Bronchiolitis Obliterans Organizing Pneumonia and Acute Respiratory Distress Syndrome 1
Article Snippet: .. Slides were then washed and incubated with M.O.M. biotinylated anti-mouse IgG reagent (1/250 dilution; Vector Laboratories) for 10 min. For IHC for caspase-8 or caspase-3, sections were incubated overnight with either a rabbit polyclonal Ab for cleaved caspase-3 (1/200 dilution; Cell Signaling Technologies) (data not shown) or a rabbit polyclonal Ab for active/cleaved caspase-8 (1/200 dilution; Imgenex) followed by incubation with a biotinylated goat anti-rabbit IgG (1/500; Santa Cruz Biotechnology) for 1 h. Immunoreactivity was demonstrated using the ABC immunostaining system kit (ABC kit; Vector Laboratories) for Fas, caspase-8, and caspase-3 or using the avidin biotin complex immunoperoxidase system from the M.O.M. immunodetection kit PK-2200 (Vector Laboratories) for FasL analysis. .. Color development was completed with the 3′,3-diaminobenzidine substrate (Sigma-Aldrich), and sections were counterstained with hematoxylin.

other:

Article Title: HECT‐Type Ubiquitin E3 Ligase ITCH Interacts With Thioredoxin‐Interacting Protein and Ameliorates Reactive Oxygen Species–Induced Cardiotoxicity
Article Snippet: Rabbit polyclonal anti–cleaved caspase‐3, rabbit polyclonal anti–precaspase‐3, rabbit polyclonal anti–cleaved caspase‐9, rabbit polyclonal anti–phospho‐p38 MAPK, anti–p38 MAPK, rabbit polyclonal anti–phospho‐p53, mouse monoclonal anti‐p53, rabbit polyclonal anti–Bcl‐2, rabbit polyclonal anti–p22‐phox and rabbit polyclonal anti–β‐tubulin antibody were purchased from Cell Signaling Corp. Anti‐FLAG antibody was purchased from Sigma‐Aldrich.

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  • 89
    Cell Signaling Technology Inc anti β catenin polyclonal antibody
    SUMOylation inhibition represses the <t>Wnt/β-catenin</t> pathway. A. For myeloma cells NCI-H929, the silencing efficiency with SUMO-1 siRNA was tested 48 hours after transfection. Total RNA was then extracted, and SUMO-1 mRNA level was determined by real time RT-PCR. Whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 antibody. SUMO-1 was indicated by arrow and was about 15 kDa in size, while SUMOylation pattern was detected as conjugated proteins with SUMO-1 modification. B. Effects of SUMO-1 inhibition on TCF/β-catenin reporter activity. Myeloma cells were transfected with control siRNA or siRNA targeting SUMO-1. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after re-transfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. Data represent mean ± S.D.; *p
    Anti β Catenin Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti β catenin polyclonal antibody/product/Cell Signaling Technology Inc
    Average 89 stars, based on 8 article reviews
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    anti β catenin polyclonal antibody - by Bioz Stars, 2020-09
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    85
    Cell Signaling Technology Inc anti phospho threonine 668 app rabbit polyclonal antibody
    ( A ) siRNA knockdown of JIP1 does not affect <t>APP</t> processing or calsyntenin-1 levels in rat cortical neurons. Neurons were treated with control (Ctrl) or JIP1 mix siRNAs (JIP1) and the samples probed on immunoblots for full-length APP (APP), APP phosphorylated on <t>threonine-668</t> (pAPP), total sAPP, sAPPα and sAPPβ in conditioned media, and calsyntenin-1 (Cstn); actin is shown as a loading control. No significant changes in the levels of any of these proteins or phosphorylation of APP on threonine-668 were detected between control or JIP1-siRNA treated neurons (Student's t -test n = 3). ( B ) siRNA knockdown of calsyntenin-1 does not affect JIP1 protein levels. Neurons were treated with control (Ctrl) or calsyntenin-1 (Cstn) siRNAs and the samples probed on immunoblots for JIP1 and actin as a loading control. No significant changes in the levels of JIP1 were detected between control or calsyntenin-1 siRNA-treated neurons (Student's t -test n = 3).
    Anti Phospho Threonine 668 App Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Cell Signaling Technology Inc rabbit anti phospho vegfr2 y1175 pab
    Necl-4 interacts with VEGFR1 and <t>VEGFR2</t> through their extracellular regions. A , Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. B , Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 <t>pAb</t> and samples were assessed by Western blotting using the indicated antibodies. C and D , Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 ( C ) or VEGFR2 ( D ) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.
    Rabbit Anti Phospho Vegfr2 Y1175 Pab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho vegfr2 y1175 pab/product/Cell Signaling Technology Inc
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    Cell Signaling Technology Inc polyclonal anti npm1 rabbit antibody
    Western blot analysis with representative sera in ELISA. Lane 1, the <t>polyclonal</t> <t>anti-NPM1</t> antibody was used as positive control. Lanes 2–4, three representative HCC sera which were positive in ELISA also had strong reactivity with NPM1 recombinant protein in western blot analysis. Lanes 5 and 6, two randomly selected NHS had negative reactivity to NPM1 recombinant protein. NPM1, nucleophosmin; HCC, hepatocellular carcinoma; NHS, normal human sera.
    Polyclonal Anti Npm1 Rabbit Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SUMOylation inhibition represses the Wnt/β-catenin pathway. A. For myeloma cells NCI-H929, the silencing efficiency with SUMO-1 siRNA was tested 48 hours after transfection. Total RNA was then extracted, and SUMO-1 mRNA level was determined by real time RT-PCR. Whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 antibody. SUMO-1 was indicated by arrow and was about 15 kDa in size, while SUMOylation pattern was detected as conjugated proteins with SUMO-1 modification. B. Effects of SUMO-1 inhibition on TCF/β-catenin reporter activity. Myeloma cells were transfected with control siRNA or siRNA targeting SUMO-1. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after re-transfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. Data represent mean ± S.D.; *p

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: SUMOylation inhibition represses the Wnt/β-catenin pathway. A. For myeloma cells NCI-H929, the silencing efficiency with SUMO-1 siRNA was tested 48 hours after transfection. Total RNA was then extracted, and SUMO-1 mRNA level was determined by real time RT-PCR. Whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 antibody. SUMO-1 was indicated by arrow and was about 15 kDa in size, while SUMOylation pattern was detected as conjugated proteins with SUMO-1 modification. B. Effects of SUMO-1 inhibition on TCF/β-catenin reporter activity. Myeloma cells were transfected with control siRNA or siRNA targeting SUMO-1. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after re-transfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. Data represent mean ± S.D.; *p

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Inhibition, Transfection, Quantitative RT-PCR, Modification, Activity Assay, Incubation, Plasmid Preparation, Negative Control, Luciferase

    Overexpression of β-catenin rescued Wnt/β-catenin pathway activity and partially prevented increased apoptosis and growth inhibition induced by SUMOylation inhibition. A. Myeloma cells NCI-H929 were treated with SUMO-1 siRNA and/or β-catenin (S33Y) plasmid for 48 hours. Protein samples were subjected to western blot analysis for the Wnt/β-catenin downstream effector proteins (c-myc, cyclinD1, survivin) with indicated antibodies. B. The distribution of apoptotic cells following treatment of myeloma cells with SUMO-1 siRNA, β-catenin (S33Y) expression vector, or both were evaluated using annexin V and propidium iodide (PI) double staining. Early apoptotic cells (Annexin-V + and PI - ) were displayed in the lower right quadrant and late apoptotic cells (Annexin-V + and PI + ) were shown in the upper right quadrant. C. The percentage of all the apoptotic cells (including Annexin-V + PI - and Annexin-V + PI + cells) were indicated by Annexin-V + cells shown as mean ± SD from three independent experiments. D. The growth inhibition of myeloma cells following different treatments was shown by the percentage of relative survival rate (OD value of sample/OD value of control in each group×100%) (*p

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: Overexpression of β-catenin rescued Wnt/β-catenin pathway activity and partially prevented increased apoptosis and growth inhibition induced by SUMOylation inhibition. A. Myeloma cells NCI-H929 were treated with SUMO-1 siRNA and/or β-catenin (S33Y) plasmid for 48 hours. Protein samples were subjected to western blot analysis for the Wnt/β-catenin downstream effector proteins (c-myc, cyclinD1, survivin) with indicated antibodies. B. The distribution of apoptotic cells following treatment of myeloma cells with SUMO-1 siRNA, β-catenin (S33Y) expression vector, or both were evaluated using annexin V and propidium iodide (PI) double staining. Early apoptotic cells (Annexin-V + and PI - ) were displayed in the lower right quadrant and late apoptotic cells (Annexin-V + and PI + ) were shown in the upper right quadrant. C. The percentage of all the apoptotic cells (including Annexin-V + PI - and Annexin-V + PI + cells) were indicated by Annexin-V + cells shown as mean ± SD from three independent experiments. D. The growth inhibition of myeloma cells following different treatments was shown by the percentage of relative survival rate (OD value of sample/OD value of control in each group×100%) (*p

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Over Expression, Activity Assay, Inhibition, Plasmid Preparation, Western Blot, Expressing, Double Staining

    SUMOylation inhibition promotes the degradation of β-catenin via the ubiquitin-proteasomal pathway. A. Myeloma cells were treated with SUMO-1 siRNA or negative control for 48 hours. Total RNA was then extracted, and β-catenin mRNA level was determined by real time RT-PCR. B. Myeloma cells NCI-H929 and RPMI-8226 were transfected with control or siRNA targeting SUMO-1. After 48 h incubation, whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. C. Myeloma cells NCI-H929 were treated with mounting doses of SUMO-1 siRNA (30 nM, 50 nM, 100 nM) for 48 h. Then whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. D. Myeloma cells were transfected with control or siRNA targeting SUMO-1. After 48 hours, cells were treated with CHX (100 μg/ml) for indicated time periods (0, 0.5, 1, 2 hours). Cell lysate was subjected to western blot with anti-β-catenin antibody. The relative intensity of β-catenin proteins at the indicated time points was quantified using SmartViewsoftware. In the negative control group, the intensity was standardized to 100% of the control sample (CHX 0 h); while in the SUMO-1 siRNA group, it was standardized to 100% of the control sample in the SUMO-1 siRNA group (CHX 0 h). This result is a representative of three independent experiments. E. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. After 48 hours, the cells were harvested under denaturing conditions and immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-ubiquitin and anti-β-catenin antibodies respectively. F. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. 48 hours after transfection, cells were treated with MG132 (10 μM) for 5 hours. The protein extracts were then analyzed by western blot using antibodies against β-catenin, SUMO-1 or actin as loading control. G. Myeloma cells were transfected with SUMO-1 siRNA together with β-catenin (WT) or β-catenin (S33Y) plasmids. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after retransfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. (Data represent mean ± S.D.; **p

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: SUMOylation inhibition promotes the degradation of β-catenin via the ubiquitin-proteasomal pathway. A. Myeloma cells were treated with SUMO-1 siRNA or negative control for 48 hours. Total RNA was then extracted, and β-catenin mRNA level was determined by real time RT-PCR. B. Myeloma cells NCI-H929 and RPMI-8226 were transfected with control or siRNA targeting SUMO-1. After 48 h incubation, whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. C. Myeloma cells NCI-H929 were treated with mounting doses of SUMO-1 siRNA (30 nM, 50 nM, 100 nM) for 48 h. Then whole cell lysate was collected and lysed and immunoblotted with anti-SUMO-1 or anti-β-catenin antibody respectively. D. Myeloma cells were transfected with control or siRNA targeting SUMO-1. After 48 hours, cells were treated with CHX (100 μg/ml) for indicated time periods (0, 0.5, 1, 2 hours). Cell lysate was subjected to western blot with anti-β-catenin antibody. The relative intensity of β-catenin proteins at the indicated time points was quantified using SmartViewsoftware. In the negative control group, the intensity was standardized to 100% of the control sample (CHX 0 h); while in the SUMO-1 siRNA group, it was standardized to 100% of the control sample in the SUMO-1 siRNA group (CHX 0 h). This result is a representative of three independent experiments. E. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. After 48 hours, the cells were harvested under denaturing conditions and immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-ubiquitin and anti-β-catenin antibodies respectively. F. Myeloma cells NCI-H929 were transfected with SUMO-1 siRNA or negative control. 48 hours after transfection, cells were treated with MG132 (10 μM) for 5 hours. The protein extracts were then analyzed by western blot using antibodies against β-catenin, SUMO-1 or actin as loading control. G. Myeloma cells were transfected with SUMO-1 siRNA together with β-catenin (WT) or β-catenin (S33Y) plasmids. After 48 h incubation, cells were transfected with TOPflash reporter plasmid or FOPflash-negative control plasmid. Twenty-four hours after retransfection, cells were harvested and luciferase activity was measured as described in Materials and Methods. All results were normalized for transfection efficiency using the hRL-Null Renilla plasmid. Fold induction corresponds to luciferase activity of positive TOPflash reporter over negative FOPflash reporter. (Data represent mean ± S.D.; **p

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Inhibition, Negative Control, Quantitative RT-PCR, Transfection, Incubation, Western Blot, Immunoprecipitation, SDS Page, Plasmid Preparation, Luciferase, Activity Assay

    Endogenous β-catenin is modified by SUMO-1 in myeloma cells. A. Myeloma cells RPMI-8226 and NCI-H929 were harvested under denaturing conditions as described under “Experimental Procedures” and immunoprecipitated with anti-β–catenin rabbit antibody or IgG as a control. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively. B. NCI-H929 was transfected with Flag-tagged β-catenin or together with His-tagged SUMO-1 or Myc-tagged SENP1, after 48 hours, cells were harvested and then subjected to western blot with anti-Flag antibody. C. Endogenous β-catenin co-localized with SUMO-1. Myeloma cells NCI-H929 were fixed and stained with anti-β-catenin mouse antibody (green) and anti-SUMO-1 rabbit antibody (red). DNA was stained with DAPI. The images were taken by confocal microscopy as described under “Experimental Procedures”. D. After transfection of negative control and/or SUMO-1 siRNA for 48 hours, whole cell lysates were immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 respectively. SUMOylated β-catenin was indicated by arrow head. E. Bone marrow samples were isolated from myeloma patients. Total lysate was prepared from 5 × 10 6 cells in each sample and immunoprecipitated with anti-β–catenin rabbit antibody as mentioned above. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively.

    Journal: American Journal of Cancer Research

    Article Title: β-catenin SUMOylation is involved in the dysregulated proliferation of myeloma cells

    doi:

    Figure Lengend Snippet: Endogenous β-catenin is modified by SUMO-1 in myeloma cells. A. Myeloma cells RPMI-8226 and NCI-H929 were harvested under denaturing conditions as described under “Experimental Procedures” and immunoprecipitated with anti-β–catenin rabbit antibody or IgG as a control. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively. B. NCI-H929 was transfected with Flag-tagged β-catenin or together with His-tagged SUMO-1 or Myc-tagged SENP1, after 48 hours, cells were harvested and then subjected to western blot with anti-Flag antibody. C. Endogenous β-catenin co-localized with SUMO-1. Myeloma cells NCI-H929 were fixed and stained with anti-β-catenin mouse antibody (green) and anti-SUMO-1 rabbit antibody (red). DNA was stained with DAPI. The images were taken by confocal microscopy as described under “Experimental Procedures”. D. After transfection of negative control and/or SUMO-1 siRNA for 48 hours, whole cell lysates were immunoprecipitated with anti-β-catenin antibody. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 respectively. SUMOylated β-catenin was indicated by arrow head. E. Bone marrow samples were isolated from myeloma patients. Total lysate was prepared from 5 × 10 6 cells in each sample and immunoprecipitated with anti-β–catenin rabbit antibody as mentioned above. The immunoprecipitates (IP) were resolved by SDS-PAGE and immunoblotted with anti-β-catenin and anti-SUMO-1 antibodies respectively. SUMOylated β-catenin and non-SUMOylated β-catenin were indicated by arrow head and arrow, respectively.

    Article Snippet: Then 20 μL was loaded onto Tricine-SDS-PAGE gels, and probed with anti-β-catenin polyclonal antibody (rabbit, CST).

    Techniques: Modification, Immunoprecipitation, SDS Page, Transfection, Western Blot, Staining, Confocal Microscopy, Negative Control, Isolation

    ( A ) siRNA knockdown of JIP1 does not affect APP processing or calsyntenin-1 levels in rat cortical neurons. Neurons were treated with control (Ctrl) or JIP1 mix siRNAs (JIP1) and the samples probed on immunoblots for full-length APP (APP), APP phosphorylated on threonine-668 (pAPP), total sAPP, sAPPα and sAPPβ in conditioned media, and calsyntenin-1 (Cstn); actin is shown as a loading control. No significant changes in the levels of any of these proteins or phosphorylation of APP on threonine-668 were detected between control or JIP1-siRNA treated neurons (Student's t -test n = 3). ( B ) siRNA knockdown of calsyntenin-1 does not affect JIP1 protein levels. Neurons were treated with control (Ctrl) or calsyntenin-1 (Cstn) siRNAs and the samples probed on immunoblots for JIP1 and actin as a loading control. No significant changes in the levels of JIP1 were detected between control or calsyntenin-1 siRNA-treated neurons (Student's t -test n = 3).

    Journal: Human Molecular Genetics

    Article Title: Loss of c-Jun N-terminal kinase-interacting protein-1 does not affect axonal transport of the amyloid precursor protein or A? production

    doi: 10.1093/hmg/ddt313

    Figure Lengend Snippet: ( A ) siRNA knockdown of JIP1 does not affect APP processing or calsyntenin-1 levels in rat cortical neurons. Neurons were treated with control (Ctrl) or JIP1 mix siRNAs (JIP1) and the samples probed on immunoblots for full-length APP (APP), APP phosphorylated on threonine-668 (pAPP), total sAPP, sAPPα and sAPPβ in conditioned media, and calsyntenin-1 (Cstn); actin is shown as a loading control. No significant changes in the levels of any of these proteins or phosphorylation of APP on threonine-668 were detected between control or JIP1-siRNA treated neurons (Student's t -test n = 3). ( B ) siRNA knockdown of calsyntenin-1 does not affect JIP1 protein levels. Neurons were treated with control (Ctrl) or calsyntenin-1 (Cstn) siRNAs and the samples probed on immunoblots for JIP1 and actin as a loading control. No significant changes in the levels of JIP1 were detected between control or calsyntenin-1 siRNA-treated neurons (Student's t -test n = 3).

    Article Snippet: Anti-APP mouse clone 22C11 was from Merck Millipore (Billerica, USA); anti-sAPPα clone 2B3 and anti-sAPPβ rabbit polyclonal antibodies were from IBL International (Hamburg, Germany), anti-phospho-threonine-668 APP rabbit polyclonal antibody was from Cell Signaling Technology (Danvers, USA).

    Techniques: Western Blot

    Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions. A , Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. B , Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. C and D , Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 ( C ) or VEGFR2 ( D ) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.

    Journal: PLoS ONE

    Article Title: The Cell Adhesion Molecule Necl-4/CADM4 Serves as a Novel Regulator for Contact Inhibition of Cell Movement and Proliferation

    doi: 10.1371/journal.pone.0124259

    Figure Lengend Snippet: Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions. A , Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. B , Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. C and D , Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 ( C ) or VEGFR2 ( D ) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.

    Article Snippet: Alexa 488-conjugated isolectin B4 (Life Technologies, Carlsbad, CA, USA), goat anti-vascular endothelial cadherin (VE-cadherin) pAb (sc-6458, Santa Cruz Biotechnology), mouse anti-Necl-4/SynCAM4 mAb (UC Davis/NIH NeuroMab Facility, Davis, CA, USA), mouse anti-human Necl-5/CD155 mAb (MAB2530, R & D Systems, Inc., Minneapolis, MN), rabbit anti-nectin-2 mAb (ab135246, Abcam, Cambridge, UK), goat anti-nectin-3 pAb (sc-14806, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-vinculin mAb (V4505, Sigma-Aldrich, St. Louis, MO, USA), Alexa 488-conjugated phalloidin (Life Technologies), mouse anti-afadin/AF6 mAb (610732, BD Biosciences, San Jose, CA, USA), rabbit anti-Rap1 pAb (sc-65, Santa Cruz Biotechnology), mouse anti-FLAG mAb (F1804, Sigma-Aldrich), rabbit anti-FLAG pAb (F7425, Sigma-Aldrich), rabbit anti-VEGF receptor (VEGFR) 1 pAb (sc-316, Santa Cruz Biotechnology), rabbit anti-phospho-VEGFR2 (Y1175) pAb (#3770, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-VEGFR2 pAb (sc-504, Santa Cruz Biotechnology), rabbit anti-p44/42 MAPK pAb (#9102, Cell Signaling Technology), rabbit anti-phospho-p44/42 MAPK (T202/Y204) pAb (#9101, Cell Signaling Technology), rabbit anti-phospho-myosin phosphatase target subunit 1 (MYPT1)/myosin-binding subunit (MBS) (Thr853) pAb (#4563, Cell Signaling Technology), rabbit anti-MYPT1/MBS pAb (#2634, Cell Signaling Technology), mouse anti-Rac1 mAb (610650, BD Biosciences), rabbit anti-PTPN13 pAb (PAB0256, Abnova, Taipei, Taiwan), and mouse anti-actin mAb (sc-8432, Santa Cruz Biotechnology; MAB1501, Merck Millipore, Billerica, MA, USA) were purchased from the indicated suppliers.

    Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Cell Culture, Immunoprecipitation

    Western blot analysis with representative sera in ELISA. Lane 1, the polyclonal anti-NPM1 antibody was used as positive control. Lanes 2–4, three representative HCC sera which were positive in ELISA also had strong reactivity with NPM1 recombinant protein in western blot analysis. Lanes 5 and 6, two randomly selected NHS had negative reactivity to NPM1 recombinant protein. NPM1, nucleophosmin; HCC, hepatocellular carcinoma; NHS, normal human sera.

    Journal: Oncology Reports

    Article Title: Humoral autoimmune response to nucleophosmin in the immunodiagnosis of hepatocellular carcinoma

    doi: 10.3892/or.2015.3854

    Figure Lengend Snippet: Western blot analysis with representative sera in ELISA. Lane 1, the polyclonal anti-NPM1 antibody was used as positive control. Lanes 2–4, three representative HCC sera which were positive in ELISA also had strong reactivity with NPM1 recombinant protein in western blot analysis. Lanes 5 and 6, two randomly selected NHS had negative reactivity to NPM1 recombinant protein. NPM1, nucleophosmin; HCC, hepatocellular carcinoma; NHS, normal human sera.

    Article Snippet: Polyclonal anti-NPM1 rabbit antibody and monoclonal anti-β-actin mouse antibody were obtained from commercial sources (Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control, Recombinant

    Representative immunofluorescence staining pattern of anti-NPM1 autoantibody-positive HCC serum. (A) NHS were used as a negative control. (B) Polyclonal anti-NPM1 antibody which showed a nuclear immunofluorescence staining pattern was used as a positive control (green signal). (C) Representative anti-NPM1 autoantibody-positive HCC serum demonstrated an intense nuclear staining pattern. (D) The same HCC serum used in panel C was pre-absorbed with recombinant NPM1. The nuclear fluorescent staining was significantly reduced. Scale bars, 20 μ m. NPM1, nucleophosmin; HCC, hepatocellular carcinoma; NHS, normal human sera. DNA was stained with DAPI (blue) (left panels).

    Journal: Oncology Reports

    Article Title: Humoral autoimmune response to nucleophosmin in the immunodiagnosis of hepatocellular carcinoma

    doi: 10.3892/or.2015.3854

    Figure Lengend Snippet: Representative immunofluorescence staining pattern of anti-NPM1 autoantibody-positive HCC serum. (A) NHS were used as a negative control. (B) Polyclonal anti-NPM1 antibody which showed a nuclear immunofluorescence staining pattern was used as a positive control (green signal). (C) Representative anti-NPM1 autoantibody-positive HCC serum demonstrated an intense nuclear staining pattern. (D) The same HCC serum used in panel C was pre-absorbed with recombinant NPM1. The nuclear fluorescent staining was significantly reduced. Scale bars, 20 μ m. NPM1, nucleophosmin; HCC, hepatocellular carcinoma; NHS, normal human sera. DNA was stained with DAPI (blue) (left panels).

    Article Snippet: Polyclonal anti-NPM1 rabbit antibody and monoclonal anti-β-actin mouse antibody were obtained from commercial sources (Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Immunofluorescence, Staining, Negative Control, Positive Control, Recombinant

    Expression of NPM1 in liver cancer and normal hepatic tissues by immunohistochemistry. The polyclonal anti-NPM1 antibody was used as a primary antibody to detect the expression of NPM1 in liver cancer and normal hepatic tissues. (A) Normal hepatic tissue with negative staining. (B) HCC tissue (grade II) with a positive staining signal. (C) HCC tissue (grade III) with a strong positive staining signal. (D) A normal hepatic tissue with H E staining signal. (E) HCC tissue (grade II) with H E staining signal. (F) HCC tissue (grade III) with H E staining signal. Grade II, moderately differentiated, cells appear slightly different than normal. Grade III, poorly differentiated, cells appear abnormal and tend to grow and spread more aggressively. NPM1, nucleophosmin; HCC, hepatocellular carcinoma; H E, hematoxylin and eosin.

    Journal: Oncology Reports

    Article Title: Humoral autoimmune response to nucleophosmin in the immunodiagnosis of hepatocellular carcinoma

    doi: 10.3892/or.2015.3854

    Figure Lengend Snippet: Expression of NPM1 in liver cancer and normal hepatic tissues by immunohistochemistry. The polyclonal anti-NPM1 antibody was used as a primary antibody to detect the expression of NPM1 in liver cancer and normal hepatic tissues. (A) Normal hepatic tissue with negative staining. (B) HCC tissue (grade II) with a positive staining signal. (C) HCC tissue (grade III) with a strong positive staining signal. (D) A normal hepatic tissue with H E staining signal. (E) HCC tissue (grade II) with H E staining signal. (F) HCC tissue (grade III) with H E staining signal. Grade II, moderately differentiated, cells appear slightly different than normal. Grade III, poorly differentiated, cells appear abnormal and tend to grow and spread more aggressively. NPM1, nucleophosmin; HCC, hepatocellular carcinoma; H E, hematoxylin and eosin.

    Article Snippet: Polyclonal anti-NPM1 rabbit antibody and monoclonal anti-β-actin mouse antibody were obtained from commercial sources (Cell Signaling Technology, Inc., Danvers, MA, USA).

    Techniques: Expressing, Immunohistochemistry, Negative Staining, Staining