antibodies against human cxcr1  (Santa Cruz Biotechnology)

 
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    Name:
    IL 8RA Antibody
    Description:
    Anti IL 8RA Antibody B 1 is a mouse monoclonal IgG1 kappa light chain IL 8RA antibody provided at 200 µg ml epitope mapping at the N terminus of IL 8RA of human origin Anti IL 8RA Antibody B 1 is recommended for detection of IL 8RA of human and hamster origin by WB IP IF IHC P and ELISA Anti IL 8RA Antibody B 1 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of IL 8RA B 1 sc 7303
    Catalog Number:
    SC-7303
    Price:
    None
    Category:
    Antibodies Primary Antibodies and ImmunoCruz Conjugates Membrane Receptors IL 8RA Antibodies IL 8RA Antibody B 1
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    Structured Review

    Santa Cruz Biotechnology antibodies against human cxcr1
    Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of <t>CXCR1</t> and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.
    Anti IL 8RA Antibody B 1 is a mouse monoclonal IgG1 kappa light chain IL 8RA antibody provided at 200 µg ml epitope mapping at the N terminus of IL 8RA of human origin Anti IL 8RA Antibody B 1 is recommended for detection of IL 8RA of human and hamster origin by WB IP IF IHC P and ELISA Anti IL 8RA Antibody B 1 is available conjugated to agarose for IP HRP for WB IHC P and ELISA and to either phycoerythrin or FITC for IF IHC P and FCM also available conjugated to Alexa Fluor 488 Alexa Fluor 546 Alexa Fluor 594 or Alexa Fluor 647 for WB RGB IF IHC P and FCM and for use with RGB fluorescent imaging systems such as iBright FL1000 FluorChem Typhoon Azure and other comparable systems also available conjugated to Alexa Fluor 680 or Alexa Fluor 790 for WB NIR IF and FCM for use with Near Infrared NIR detection systems such as LI COR Odyssey iBright FL1000 FluorChem Typhoon Azure and other comparable systems Contact our Technical Service Department or your local Distributor for more information on how to receive a FREE 10 µg sample of IL 8RA B 1 sc 7303
    https://www.bioz.com/result/antibodies against human cxcr1/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against human cxcr1 - by Bioz Stars, 2021-09
    92/100 stars

    Images

    1) Product Images from "Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy"

    Article Title: Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0201858

    Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of CXCR1 and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.
    Figure Legend Snippet: Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of CXCR1 and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Western Blot, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Proliferation Assay

    Related Articles

    Immunofluorescence:

    Article Title: Effect of cold and hot compress on neutrophilic migration to the site of doxorubicin extravasation
    Article Snippet: .. We then fixed the sections with cold acetone (4°C) for 10 min, followed by addition of 3% skim milk (Morinaga Milk Industry, Tokyo, Japan) dissolved in PBS after washing in 0.01 M phosphate-buffered saline, pH 7.4 (PBS), for 15 min. For double immunofluorescence, slide-mounted tissue sections were incubated with a primary antibody [anti-CD88 (C5aR) antibody (rabbit, polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA), anti-rat IL-8RA antibody (recognizing mouse IL-8RA, goat polyclonal, Santa Cruz Biotechnology), anti-rat TRPV1 (recognizing mouse TRPV1, rabbit, polyclonal, Alomone Labs, Jerusalem, Israel) and mouse monoclonal anti-α-SMA antibody (Clone 1A4, mouse IgG2a, Dako, Glostrup, Denmark)] overnight at 4°C. ..

    Incubation:

    Article Title: Effect of cold and hot compress on neutrophilic migration to the site of doxorubicin extravasation
    Article Snippet: .. We then fixed the sections with cold acetone (4°C) for 10 min, followed by addition of 3% skim milk (Morinaga Milk Industry, Tokyo, Japan) dissolved in PBS after washing in 0.01 M phosphate-buffered saline, pH 7.4 (PBS), for 15 min. For double immunofluorescence, slide-mounted tissue sections were incubated with a primary antibody [anti-CD88 (C5aR) antibody (rabbit, polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA), anti-rat IL-8RA antibody (recognizing mouse IL-8RA, goat polyclonal, Santa Cruz Biotechnology), anti-rat TRPV1 (recognizing mouse TRPV1, rabbit, polyclonal, Alomone Labs, Jerusalem, Israel) and mouse monoclonal anti-α-SMA antibody (Clone 1A4, mouse IgG2a, Dako, Glostrup, Denmark)] overnight at 4°C. ..

    other:

    Article Title: Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy
    Article Snippet: Antibodies against human CXCR1 (sc-7303), CXCR2 (sc-7304), IKKα (sc-7218), IKKβ (sc-8014), IKKε (sc-376114), p65 NFκB (sc-372), IκBα (sc-371), and histone H3 (sc-8654) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Article Title: Histone Deacetylase (HDAC) Inhibition Induces IκB Kinase (IKK)-dependent Interleukin-8/CXCL8 Expression in Ovarian Cancer Cells *
    Article Snippet: The antibodies against human p65 NFκB (sc-372), p50 NFκB (sc-7178), IκBα (sc-371), histone H3 (sc-8654), Lys-9/14-acetylated histone H3 (sc-8655), IKKα (sc-7218), IKKβ (sc-8014), IKKϵ (sc-376114), IL-8/CXCL8 (sc-7922), CXCR1 (sc-7303), and CXCR2 (sc-7304) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

    Article Title: Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy
    Article Snippet: Antibodies and reagents Antibodies against human CXCR1 (sc-7303), CXCR2 (sc-7304), IKKα (sc-7218), IKKβ (sc-8014), IKKε (sc-376114), p65 NFκB (sc-372), IκBα (sc-371), and histone H3 (sc-8654) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Expressing:

    Article Title: IL-1?-INDUCED POST-TRANSITION EFFECT OF NF-KAPPAB PROVIDES TIME-DEPENDENT WAVE OF SIGNALS FOR INITIAL PHASE OF INTRAPOSTATIC INFLAMMATION
    Article Snippet: .. The intraprostatic expression of chemokine receptors, adhesion molecules, surface cell markers, and interleukin in formalin-fixed paraffin-embedded prostate tissue was evaluated using the following markers: for chemokine receptors - CXCR1/IL-8RA (Santa Cruz Biotechnology, Santa Cruz, CA) and CXCR4 (R & D System, Minneapolis, MN); for adhesion molecules - VCAM1 (Santa Cruz); for neutrophils - NIMP-R14 (Santa Cruz); for mononuclear cells (MNC) - CD45 (R & D System); and for IL-17F - (R & D System). ..

    Formalin-fixed Paraffin-Embedded:

    Article Title: IL-1?-INDUCED POST-TRANSITION EFFECT OF NF-KAPPAB PROVIDES TIME-DEPENDENT WAVE OF SIGNALS FOR INITIAL PHASE OF INTRAPOSTATIC INFLAMMATION
    Article Snippet: .. The intraprostatic expression of chemokine receptors, adhesion molecules, surface cell markers, and interleukin in formalin-fixed paraffin-embedded prostate tissue was evaluated using the following markers: for chemokine receptors - CXCR1/IL-8RA (Santa Cruz Biotechnology, Santa Cruz, CA) and CXCR4 (R & D System, Minneapolis, MN); for adhesion molecules - VCAM1 (Santa Cruz); for neutrophils - NIMP-R14 (Santa Cruz); for mononuclear cells (MNC) - CD45 (R & D System); and for IL-17F - (R & D System). ..

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    Santa Cruz Biotechnology goat anti human cxcr2
    Staphopain A inhibits calcium mobilization of <t>U937-CXCR2</t> cells. Fluo-3-labelled U937-CXCR2 cells were preincubated with buffer, or 0.5 μM Staphopain A with and without 1 μM Staphostatin A for 15 min at 37°C. After washing, cells were stimulated with different concentrations of CXCL1 ( A ), CXCL7 ( B ) and CXCL8 ( C ). All figures represent the mean±s.e. of three separate experiments. The relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. * P
    Goat Anti Human Cxcr2, supplied by Santa Cruz Biotechnology, 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/goat anti human cxcr2/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti human cxcr2 - by Bioz Stars, 2021-09
    86/100 stars
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    92
    Santa Cruz Biotechnology antibodies against human cxcr1
    Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of <t>CXCR1</t> and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.
    Antibodies Against Human Cxcr1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against human cxcr1/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against human cxcr1 - by Bioz Stars, 2021-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    Staphopain A inhibits calcium mobilization of U937-CXCR2 cells. Fluo-3-labelled U937-CXCR2 cells were preincubated with buffer, or 0.5 μM Staphopain A with and without 1 μM Staphostatin A for 15 min at 37°C. After washing, cells were stimulated with different concentrations of CXCL1 ( A ), CXCL7 ( B ) and CXCL8 ( C ). All figures represent the mean±s.e. of three separate experiments. The relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. * P

    Journal: The EMBO Journal

    Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

    doi: 10.1038/emboj.2012.212

    Figure Lengend Snippet: Staphopain A inhibits calcium mobilization of U937-CXCR2 cells. Fluo-3-labelled U937-CXCR2 cells were preincubated with buffer, or 0.5 μM Staphopain A with and without 1 μM Staphostatin A for 15 min at 37°C. After washing, cells were stimulated with different concentrations of CXCL1 ( A ), CXCL7 ( B ) and CXCL8 ( C ). All figures represent the mean±s.e. of three separate experiments. The relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. * P

    Article Snippet: After washing, cells were incubated with mouse anti-human CXCR2 (clone 19, Abcam, directed against the N-terminus), goat anti-human CXCR2 (sc-22661, Santa Cruz, directed against the extracellular domain) or mouse anti-human CXCR1 (R & D, directed against the N-terminus) for 30 min on ice.

    Techniques: Fluorescence

    Staphopain A inhibits CXCR2-mediated calcium mobilization of neutrophils. Fluo-3-labelled neutrophils were preincubated with buffer or 0.5 μM Staphopain A with or without 1 μM Staphostatin A, for 15 min at 37°C. After washing, cells were stimulated with different concentrations of CXCL1 ( A ), CXCL7 ( B ), CXCL8 ( C ), fMLF ( D ), C5a ( E ) or a fixed concentration (3 × 10 −9 M) of CXCL2, CXCL3, CXCL5 and CXCL6 ( F ). To determine the IC 50 (50% of inhibition), Fluo-3-labelled neutrophils were preincubated with different concentrations of Staphopain A for 15 min at 37°C and subsequently stimulated with 3 × 10 −9 M CXCL1 ( G ) or 1 × 10 −8 M CXCL7 ( H ). The IC 50 for CXCL1 and CXCL7 was calculated with the formulas y =−4 E +10 −7 x +4.1314 and y =−3 E +10 −7 x +4.0248, respectively. All figures represent the mean±s.e. of three separate experiments using different donors. The relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. * P

    Journal: The EMBO Journal

    Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

    doi: 10.1038/emboj.2012.212

    Figure Lengend Snippet: Staphopain A inhibits CXCR2-mediated calcium mobilization of neutrophils. Fluo-3-labelled neutrophils were preincubated with buffer or 0.5 μM Staphopain A with or without 1 μM Staphostatin A, for 15 min at 37°C. After washing, cells were stimulated with different concentrations of CXCL1 ( A ), CXCL7 ( B ), CXCL8 ( C ), fMLF ( D ), C5a ( E ) or a fixed concentration (3 × 10 −9 M) of CXCL2, CXCL3, CXCL5 and CXCL6 ( F ). To determine the IC 50 (50% of inhibition), Fluo-3-labelled neutrophils were preincubated with different concentrations of Staphopain A for 15 min at 37°C and subsequently stimulated with 3 × 10 −9 M CXCL1 ( G ) or 1 × 10 −8 M CXCL7 ( H ). The IC 50 for CXCL1 and CXCL7 was calculated with the formulas y =−4 E +10 −7 x +4.1314 and y =−3 E +10 −7 x +4.0248, respectively. All figures represent the mean±s.e. of three separate experiments using different donors. The relative calcium mobilization was calculated by dividing the fluorescence after stimulation by the baseline fluorescence. * P

    Article Snippet: After washing, cells were incubated with mouse anti-human CXCR2 (clone 19, Abcam, directed against the N-terminus), goat anti-human CXCR2 (sc-22661, Santa Cruz, directed against the extracellular domain) or mouse anti-human CXCR1 (R & D, directed against the N-terminus) for 30 min on ice.

    Techniques: Concentration Assay, Inhibition, Fluorescence

    Staphopain A specifically cleaves the N-terminus of CXCR2. ( A ) Western blot analysis of U937-CXCR2 cells incubated with buffer, 0.5 μM Staphopain A, 0.5 μM Staphopain A plus 1 μM Staphostatin A for 15 min at 37°C. Whole-cell lysates were analysed by western blotting using an mAb against the N-terminus of CXCR2. Figure represents three independent experiments. ( B ) Confocal images of HEK cells transfected with human CXCR1 or CXCR2 fused to a C-terminal YFP tag. Cells were incubated with buffer or 0.5 μM Staphopain A for 30 min at 37°C. The N-termini were stained with mAbs and subsequent Alexa633-labelled anti-mouse antibodies. YFP was artificially coloured green. Images are representatives of three independent experiments. ( C ) Flow cytometry analysis of human neutrophils treated with 0.5 μM Staphopain A or 40 nM PMA for 15 min at 37°C. Cells were first stained with antibodies against the N-terminus of CXCR2, the extracellular loop of CXCR2 or the N-terminus of CXCR1 and subsequently with FITC-labelled secondary antibodies. Antibody binding is expressed in percentages, calculated by dividing the fluorescence of treated cells by fluorescence of buffer-treated cells and multiplying with 100. Figure represents the mean±s.e. of three separate experiments using different donors. ** P

    Journal: The EMBO Journal

    Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

    doi: 10.1038/emboj.2012.212

    Figure Lengend Snippet: Staphopain A specifically cleaves the N-terminus of CXCR2. ( A ) Western blot analysis of U937-CXCR2 cells incubated with buffer, 0.5 μM Staphopain A, 0.5 μM Staphopain A plus 1 μM Staphostatin A for 15 min at 37°C. Whole-cell lysates were analysed by western blotting using an mAb against the N-terminus of CXCR2. Figure represents three independent experiments. ( B ) Confocal images of HEK cells transfected with human CXCR1 or CXCR2 fused to a C-terminal YFP tag. Cells were incubated with buffer or 0.5 μM Staphopain A for 30 min at 37°C. The N-termini were stained with mAbs and subsequent Alexa633-labelled anti-mouse antibodies. YFP was artificially coloured green. Images are representatives of three independent experiments. ( C ) Flow cytometry analysis of human neutrophils treated with 0.5 μM Staphopain A or 40 nM PMA for 15 min at 37°C. Cells were first stained with antibodies against the N-terminus of CXCR2, the extracellular loop of CXCR2 or the N-terminus of CXCR1 and subsequently with FITC-labelled secondary antibodies. Antibody binding is expressed in percentages, calculated by dividing the fluorescence of treated cells by fluorescence of buffer-treated cells and multiplying with 100. Figure represents the mean±s.e. of three separate experiments using different donors. ** P

    Article Snippet: After washing, cells were incubated with mouse anti-human CXCR2 (clone 19, Abcam, directed against the N-terminus), goat anti-human CXCR2 (sc-22661, Santa Cruz, directed against the extracellular domain) or mouse anti-human CXCR1 (R & D, directed against the N-terminus) for 30 min on ice.

    Techniques: Western Blot, Incubation, Transfection, Staining, Flow Cytometry, Cytometry, Binding Assay, Fluorescence

    Determination of the Staphopain A cleavage site in CXCR2. ( A ) Staphopain A cleaves the CXCR2 1–48 -GB1-His protein. Different concentrations of Staphopain A were incubated with 6.75 μM CXCR2 1–48 -GB1-His protein for 15 min at 37°C. Samples were analysed by SDS–PAGE and Instant blue staining. Lane 6: CXCR2 1–48 -GB1-His plus 150 nM Staphopain A plus 1 μM Staphostatin A; lane 7: Staphostatin A alone. ( B ) N-terminal sequencing of the CXCR2 cleavage product generated by incubation of 6.75 μM CXCR2 1–48 -GB1-His protein with 150 nM Staphopain A for 15 min at 37°C. Sequencing was performed once. ( C ) Staphopain A cleavage of CXCR2 1–48 -GB1-His (left, LLDA) or CXCR2 1–48 -GB1-His mutated with a Proline at position 34 (right, LPDA). CXCR2 protein at 6.75 μM, 15 min at 37°C; proteins were stained using Instant Blue. ( D ) Staphopain A cleaves CXCR2 in the presence of plasma. CXCR2 1–48 -GB1-His (1.5 μM) was incubated with Staphopain A with or without 10% human plasma for 30 min at 37°C. CXCR2 was visualized by immunoblotting using anti-CXCR2 (N-terminus specific) antibodies. ( A , C and D ) Representatives of three separate experiments are shown.

    Journal: The EMBO Journal

    Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

    doi: 10.1038/emboj.2012.212

    Figure Lengend Snippet: Determination of the Staphopain A cleavage site in CXCR2. ( A ) Staphopain A cleaves the CXCR2 1–48 -GB1-His protein. Different concentrations of Staphopain A were incubated with 6.75 μM CXCR2 1–48 -GB1-His protein for 15 min at 37°C. Samples were analysed by SDS–PAGE and Instant blue staining. Lane 6: CXCR2 1–48 -GB1-His plus 150 nM Staphopain A plus 1 μM Staphostatin A; lane 7: Staphostatin A alone. ( B ) N-terminal sequencing of the CXCR2 cleavage product generated by incubation of 6.75 μM CXCR2 1–48 -GB1-His protein with 150 nM Staphopain A for 15 min at 37°C. Sequencing was performed once. ( C ) Staphopain A cleavage of CXCR2 1–48 -GB1-His (left, LLDA) or CXCR2 1–48 -GB1-His mutated with a Proline at position 34 (right, LPDA). CXCR2 protein at 6.75 μM, 15 min at 37°C; proteins were stained using Instant Blue. ( D ) Staphopain A cleaves CXCR2 in the presence of plasma. CXCR2 1–48 -GB1-His (1.5 μM) was incubated with Staphopain A with or without 10% human plasma for 30 min at 37°C. CXCR2 was visualized by immunoblotting using anti-CXCR2 (N-terminus specific) antibodies. ( A , C and D ) Representatives of three separate experiments are shown.

    Article Snippet: After washing, cells were incubated with mouse anti-human CXCR2 (clone 19, Abcam, directed against the N-terminus), goat anti-human CXCR2 (sc-22661, Santa Cruz, directed against the extracellular domain) or mouse anti-human CXCR1 (R & D, directed against the N-terminus) for 30 min on ice.

    Techniques: Incubation, SDS Page, Staining, Sequencing, Generated

    Staphopain A inhibits antibody binding to CXCR2 on neutrophils. ( A ) Neutrophils were incubated with 0.5 μM Staphopain A or buffer for 15 min at 37°C. After washing, cells were stained with a panel of blocking antibodies against surface-expressed receptors. The relative expression was calculated by dividing the mean fluorescence of Staphopain-treated cells by Buffer-treated cells. ( B ) Staphopain A blocks antibody binding in a dose-dependent manner. Neutrophils were incubated with various concentrations of Staphopain A for 15 min at 37°C and stained with an antibody recognizing the N-terminal domain of human CXCR2. Inlay: Representative histogram of the CXCR2 expression on neutrophils treated with 0.5 μM Staphopain A compared to buffer. ( C ) Protease activity is required for antibody inhibition. Neutrophils were treated with 0.5 μM Staphopain A with or without 1 μM Staphostatin A or 10 μM E64. All figures represent the mean±s.e. of three separate experiments using different donors. Background was determined using an isotype control. ( A , B ) * P

    Journal: The EMBO Journal

    Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

    doi: 10.1038/emboj.2012.212

    Figure Lengend Snippet: Staphopain A inhibits antibody binding to CXCR2 on neutrophils. ( A ) Neutrophils were incubated with 0.5 μM Staphopain A or buffer for 15 min at 37°C. After washing, cells were stained with a panel of blocking antibodies against surface-expressed receptors. The relative expression was calculated by dividing the mean fluorescence of Staphopain-treated cells by Buffer-treated cells. ( B ) Staphopain A blocks antibody binding in a dose-dependent manner. Neutrophils were incubated with various concentrations of Staphopain A for 15 min at 37°C and stained with an antibody recognizing the N-terminal domain of human CXCR2. Inlay: Representative histogram of the CXCR2 expression on neutrophils treated with 0.5 μM Staphopain A compared to buffer. ( C ) Protease activity is required for antibody inhibition. Neutrophils were treated with 0.5 μM Staphopain A with or without 1 μM Staphostatin A or 10 μM E64. All figures represent the mean±s.e. of three separate experiments using different donors. Background was determined using an isotype control. ( A , B ) * P

    Article Snippet: After washing, cells were incubated with mouse anti-human CXCR2 (clone 19, Abcam, directed against the N-terminus), goat anti-human CXCR2 (sc-22661, Santa Cruz, directed against the extracellular domain) or mouse anti-human CXCR1 (R & D, directed against the N-terminus) for 30 min on ice.

    Techniques: Binding Assay, Incubation, Staining, Blocking Assay, Expressing, Fluorescence, Activity Assay, Inhibition

    Staphopain A detection in bacterial supernatants. ( A ) S. aureus strain USA300 (WT) and its isogenic mutant (Δ scpA ) were grown in TSB media and supernatants were collected at different time points. Above: OD 600 measurements taken at different time points; Below: cleavage of 1 μM CXCR2 1–48 -GB1-His protein by undiluted supernatants collected at different points (incubation for 30 min at 37°C unless otherwise noted). The CXCR2 1–48 -GB1-His protein was detected using western blotting with an antibody against the His tag. ( B ) Time-dependent cleavage of CXCR2 1–48 -GB1-His protein (1 μM) by 3 h supernatants (undiluted) of USA300 (WT), its isogenic mutant (Δ scpA ) and a complemented mutant (Δ scpA Comp). ( C ) CXCR2 cleavage by WT supernatant is inhibited by Staphostatin A (MBP-ScpB). CXCR2 1–48 -GB1-His protein (1 μM) was incubated with 3 h supernatants (undiluted) of USA300 (WT) in the presence of various concentrations of MBP-tagged ScpB. MBP is maltose binding protein. ( A – C) Representative blots of three separate experiments are shown.

    Journal: The EMBO Journal

    Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

    doi: 10.1038/emboj.2012.212

    Figure Lengend Snippet: Staphopain A detection in bacterial supernatants. ( A ) S. aureus strain USA300 (WT) and its isogenic mutant (Δ scpA ) were grown in TSB media and supernatants were collected at different time points. Above: OD 600 measurements taken at different time points; Below: cleavage of 1 μM CXCR2 1–48 -GB1-His protein by undiluted supernatants collected at different points (incubation for 30 min at 37°C unless otherwise noted). The CXCR2 1–48 -GB1-His protein was detected using western blotting with an antibody against the His tag. ( B ) Time-dependent cleavage of CXCR2 1–48 -GB1-His protein (1 μM) by 3 h supernatants (undiluted) of USA300 (WT), its isogenic mutant (Δ scpA ) and a complemented mutant (Δ scpA Comp). ( C ) CXCR2 cleavage by WT supernatant is inhibited by Staphostatin A (MBP-ScpB). CXCR2 1–48 -GB1-His protein (1 μM) was incubated with 3 h supernatants (undiluted) of USA300 (WT) in the presence of various concentrations of MBP-tagged ScpB. MBP is maltose binding protein. ( A – C) Representative blots of three separate experiments are shown.

    Article Snippet: After washing, cells were incubated with mouse anti-human CXCR2 (clone 19, Abcam, directed against the N-terminus), goat anti-human CXCR2 (sc-22661, Santa Cruz, directed against the extracellular domain) or mouse anti-human CXCR1 (R & D, directed against the N-terminus) for 30 min on ice.

    Techniques: Mutagenesis, Incubation, Western Blot, Binding Assay

    Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of CXCR1 and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.

    Journal: PLoS ONE

    Article Title: Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy

    doi: 10.1371/journal.pone.0201858

    Figure Lengend Snippet: Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of CXCR1 and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.

    Article Snippet: Antibodies against human CXCR1 (sc-7303), CXCR2 (sc-7304), IKKα (sc-7218), IKKβ (sc-8014), IKKε (sc-376114), p65 NFκB (sc-372), IκBα (sc-371), and histone H3 (sc-8654) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Western Blot, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Proliferation Assay

    Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of CXCR1 and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.

    Journal: PLoS ONE

    Article Title: Proteasome inhibition induces IKK-dependent interleukin-8 expression in triple negative breast cancer cells: Opportunity for combination therapy

    doi: 10.1371/journal.pone.0201858

    Figure Lengend Snippet: Suppression of BZ-induced IL-8 augments BZ cytotoxic and anti-proliferative effect in TNBC cells. (A) RT-PCR of CXCR1 and CXCR2 mRNA levels in MDA-MB-231 cells treated with increasing BZ for 24 h. (B) Western analysis of CXCR1, CXCR2, and control actin protein levels in whole cell extracts (WCE) of MDA-MB-231 cells treated with increasing BZ for 24 h. The bottom panel represents densitometric evaluation of CXCR1 and CXCR2 protein levels shown in the top panel. The CXCR1/2 densities were normalized to actin, and expressed relative to untreated cells. (C) RT-PCR of IL-8 mRNA in MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (D) IL-8 release measured by ELISA in supernatant of MDA-MB-231 cells transfected with control or IL-8 siRNA and incubated 24 h with increasing BZ. (E) Viability of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with increasing BZ, measured be Trypan Blue exclusion. (F) Proliferation of MDA-MB-231 cells transfected with control or IL-8 siRNA, and incubated 24 h with BZ, measured by CellTiter 96 One Solution Cell Proliferation Assay. (G) Viability of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. (H) Proliferation of MDA-MB-231 cells incubated 24 h with increasing BZ in the presence of control pre-immune IgG or IL-8 neutralizing monoclonal antibody. The values represent the mean +/− SE of four experiments; asterisks denote a statistically significant change compared to control.

    Article Snippet: Antibodies and reagents Antibodies against human CXCR1 (sc-7303), CXCR2 (sc-7304), IKKα (sc-7218), IKKβ (sc-8014), IKKε (sc-376114), p65 NFκB (sc-372), IκBα (sc-371), and histone H3 (sc-8654) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Multiple Displacement Amplification, Western Blot, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Proliferation Assay