Structured Review

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

Images

1) Product Images from "Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis"

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

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.212

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Mutagenesis, Incubation, Western Blot, Binding Assay

2) Product Images from "Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis"

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

Journal: The EMBO Journal

doi: 10.1038/emboj.2012.212

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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Mutagenesis, Incubation, Western Blot, Binding Assay

Related Articles

Staining:

Article Title: Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis
Article Snippet: .. Cells were washed and stained with mouse anti-human CXCR2 (clone 19, Abcam), goat anti-human CXCR2 (sc-22661, Santa Cruz) or mouse anti-human CXCR1 (R & D). ..

Article Title: Monoclonal Antibody against CXCL1 (HL2401) as a Novel Agent in Suppressing IL6 Expression and Tumoral Growth
Article Snippet: .. Proteins were transferred to Immun-Blot PVDF Membrane (Bio-Rad Laboratories) and stained using a goat anti-human CXCL1 antibody (sc-1374, dilution 1/250; Santa Cruz Biotechnology), mouse anti-human CXCR2 antibody (ab24963, dilution 1:500; Abcam), rabbit anti-human IL6 antibody (sc-1265, dilution1:200, Santa Cruz Biotechnology), goat anti-human TIMP4 antibody (sc-9375, dilution 1:200, Santa Cruz Biotechnology) and mouse anti-human β-actin antibody (AC-15, dilution 1:10,000, Sigma-Aldrich). ..

Co-Culture Assay:

Article Title: Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways
Article Snippet: .. To detect specific exchanged molecules, 1:100 dilutions of antibodies against human leukocyte antigen (HLA) class-I, class-II, CD11b, LFA-1, or CXCR1 (all from Santa Cruz Biotechnology, CA, USA), were pre-reacted with one cell type, before co-culture with a second cell type for 1 h. FITC-conjugated goat anti-mouse IgG (diluted 1:2000) was used as a secondary antibody. ..

Incubation:

Article Title: Downregulation of CXCR1 and CXCR2 Expression on Human Neutrophils by Helicobacter pylori: a New Pathomechanism in H. pylori Infection?
Article Snippet: .. The blotted membranes were blocked with phosphate-buffered saline containing 0.1% (vol/vol) Tween 20 and 3% (wt/vol) low-fat milk powder, followed by a 1-h incubation with the primary antibodies polyclonal rabbit anti-human CXCR1 and CXCR2 (Santa Cruz, Heidelberg, Germany) at a dilution of 1:200. ..

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

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    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
      Buy from Supplier

    86
    Santa Cruz Biotechnology polyclonal goat anti human cxcr 4
    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
    Polyclonal Goat Anti Human Cxcr 4, 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/polyclonal goat anti human cxcr 4/product/Santa Cruz Biotechnology
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal goat anti human cxcr 4 - by Bioz Stars, 2021-09
    86/100 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