t2 5 monoclonal anti tlr2 ab  (Hycult Biotech)


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    Hycult Biotech t2 5 monoclonal anti tlr2 ab
    T2 5 Monoclonal Anti Tlr2 Ab, supplied by Hycult Biotech, 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/t2 5 monoclonal anti tlr2 ab/product/Hycult Biotech
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    mouse monoclonal anti tlr2 r d systems cat  (R&D Systems)


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    R&D Systems mouse monoclonal anti tlr2 r d systems cat
    Mouse Monoclonal Anti Tlr2 R D Systems Cat, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Structured Review

    Santa Cruz Biotechnology monoclonal mouse anti human tlr2 antibody
    <t>TLR2</t> and TLR4 were expressed on HK-2 cells and interacted with AGE-LDL. (A) Protein expression of TLR2 and TLR4 on the surface of HK-2 cells was detected by flow cytometry analysis. (B) Co-immunoprecipitation of AGE-LDL and TLR2/4 in HK-2 cells. HK-2 cells were treated with AGE-LDL, native LDL, or AGE-BSA, respectively, the interaction between AGE-LDL and TLR2 or TLR4 was analyzed by immunoprecipitation using anti-AGE antibody and detected by immunoblotting using anti-TLR2 or anti-TLR4 antibody, respectively. The protein level of TLR2 and TLR4 in HK-2 cell lysate was examined by western blot.
    Monoclonal Mouse Anti Human Tlr2 Antibody, 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
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    Average 86 stars, based on 1 article reviews
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    1) Product Images from "AGE-LDL Activates Toll Like Receptor 4 Pathway and Promotes Inflammatory Cytokines Production in Renal Tubular Epithelial Cells"

    Article Title: AGE-LDL Activates Toll Like Receptor 4 Pathway and Promotes Inflammatory Cytokines Production in Renal Tubular Epithelial Cells

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.5246

    TLR2 and TLR4 were expressed on HK-2 cells and interacted with AGE-LDL. (A) Protein expression of TLR2 and TLR4 on the surface of HK-2 cells was detected by flow cytometry analysis. (B) Co-immunoprecipitation of AGE-LDL and TLR2/4 in HK-2 cells. HK-2 cells were treated with AGE-LDL, native LDL, or AGE-BSA, respectively, the interaction between AGE-LDL and TLR2 or TLR4 was analyzed by immunoprecipitation using anti-AGE antibody and detected by immunoblotting using anti-TLR2 or anti-TLR4 antibody, respectively. The protein level of TLR2 and TLR4 in HK-2 cell lysate was examined by western blot.
    Figure Legend Snippet: TLR2 and TLR4 were expressed on HK-2 cells and interacted with AGE-LDL. (A) Protein expression of TLR2 and TLR4 on the surface of HK-2 cells was detected by flow cytometry analysis. (B) Co-immunoprecipitation of AGE-LDL and TLR2/4 in HK-2 cells. HK-2 cells were treated with AGE-LDL, native LDL, or AGE-BSA, respectively, the interaction between AGE-LDL and TLR2 or TLR4 was analyzed by immunoprecipitation using anti-AGE antibody and detected by immunoblotting using anti-TLR2 or anti-TLR4 antibody, respectively. The protein level of TLR2 and TLR4 in HK-2 cell lysate was examined by western blot.

    Techniques Used: Expressing, Flow Cytometry, Immunoprecipitation, Western Blot

    AGE-LDL induced IL-6 and IL-8 production was mainly mediated by TLR4. (A) HK-2 cells were transfected with TLR2 siRNA, TLR4 siRNA or scramble siRNA, respectively. Western blot was performed to analyze the expression of endogenous TLR2/4 in HK-2 cells. GAPDH was used to verify equivalent loading. The effect of TLR2/4 siRNA on AGE-LDL-induced IL-6 and IL-8 expression was determined by real-time PCR (B, D) and ELISA (C, E). * P <0.05 versus untreated cells. # P <0.05 versus scramble siRNA transfected cells. Data are expressed as mean±SD of three independent experiments.
    Figure Legend Snippet: AGE-LDL induced IL-6 and IL-8 production was mainly mediated by TLR4. (A) HK-2 cells were transfected with TLR2 siRNA, TLR4 siRNA or scramble siRNA, respectively. Western blot was performed to analyze the expression of endogenous TLR2/4 in HK-2 cells. GAPDH was used to verify equivalent loading. The effect of TLR2/4 siRNA on AGE-LDL-induced IL-6 and IL-8 expression was determined by real-time PCR (B, D) and ELISA (C, E). * P <0.05 versus untreated cells. # P <0.05 versus scramble siRNA transfected cells. Data are expressed as mean±SD of three independent experiments.

    Techniques Used: Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    mouse anti human mabs against tlr2  (Thermo Fisher)


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    Thermo Fisher mouse anti human mabs against tlr2
    HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 <t>(TLR2/6;</t> 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.
    Mouse Anti Human Mabs Against Tlr2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists"

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068701

    HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 (TLR2/6; 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.
    Figure Legend Snippet: HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 (TLR2/6; 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.

    Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

    ( A–E ) HASMCs were stained intracellularly with Abs against TLR2, TLR3, TLR4, TLR7 and NOD1 (open histograms) or appropriate isotype control (shaded histograms) and analyzed by flow cytometry. Data show one out of three independent experiments. ( F ) Data are presented as relative mean fluorescence intensity (rMFI = MFI Ab /MFI isotype control ) and depicted as mean ± SEM (n = 3). ( G ) Immunocytochemistry of HASMCs stained with Abs against TLR2, ( H ) TLR3 (diluted 1∶10), ( I ) TLR4, ( J ) TLR7, ( K ) NOD1 (diluted 1∶50), and ( L ) N-series negative control reagent (diluted 1∶10). Slides were visualized using 3,3′-diaminobenzidine (brown). All slides were counterstained with hematoxylin (blue) and analyzed by microscopy; magnification 200×.
    Figure Legend Snippet: ( A–E ) HASMCs were stained intracellularly with Abs against TLR2, TLR3, TLR4, TLR7 and NOD1 (open histograms) or appropriate isotype control (shaded histograms) and analyzed by flow cytometry. Data show one out of three independent experiments. ( F ) Data are presented as relative mean fluorescence intensity (rMFI = MFI Ab /MFI isotype control ) and depicted as mean ± SEM (n = 3). ( G ) Immunocytochemistry of HASMCs stained with Abs against TLR2, ( H ) TLR3 (diluted 1∶10), ( I ) TLR4, ( J ) TLR7, ( K ) NOD1 (diluted 1∶50), and ( L ) N-series negative control reagent (diluted 1∶10). Slides were visualized using 3,3′-diaminobenzidine (brown). All slides were counterstained with hematoxylin (blue) and analyzed by microscopy; magnification 200×.

    Techniques Used: Staining, Flow Cytometry, Fluorescence, Immunocytochemistry, Negative Control, Microscopy

    mouse anti human tlr2 mab  (Hycult Biotech)


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    Hycult Biotech mouse anti human tlr2 mab
    ( A ) <t>TLR2</t> expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.
    Mouse Anti Human Tlr2 Mab, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells"

    Article Title: miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068296

    ( A ) TLR2 expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.
    Figure Legend Snippet: ( A ) TLR2 expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunofluorescence, Staining

    mouse monoclonal anti human tlr2 antibody tl2 1 conjugated to fitc  (Santa Cruz Biotechnology)

     
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    Santa Cruz Biotechnology mouse monoclonal anti human tlr2 antibody tl2 1 conjugated to fitc
    Mouse Monoclonal Anti Human Tlr2 Antibody Tl2 1 Conjugated To Fitc, 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
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    monoclonal mouse anti human tlr2 alexa fluor 488  (Becton Dickinson)

     
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    Becton Dickinson monoclonal mouse anti human tlr2 alexa fluor 488
    Monoclonal Mouse Anti Human Tlr2 Alexa Fluor 488, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    functional graded monoclonal mouse anti human tlr2  (Thermo Fisher)


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    Thermo Fisher functional graded monoclonal mouse anti human tlr2
    The effect of <t>TLR2-neutralizing</t> antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) <t>TLR2-independent</t> ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 10 6 cell/mL were pretreated with 10 μg/mL of <t>anti-TLR2,</t> anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in C and as mean ± SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.
    Functional Graded Monoclonal Mouse Anti Human Tlr2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells"

    Article Title: Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms222111560

    The effect of TLR2-neutralizing antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-independent ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in C and as mean ± SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.
    Figure Legend Snippet: The effect of TLR2-neutralizing antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-independent ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in C and as mean ± SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.

    Techniques Used: Derivative Assay, Fluorescence, Flow Cytometry

    Involvement of TLR2 and PTX-sensitive heterotrimeric G proteins in HP-NAP-induced secretion of IL-8 by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-dependent secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in . Data are expressed as mean ± SD of four independent experiments. ( B ) TLR2-dependent secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. ( C ) PTX-sensitive secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP and Pam 3 CSK 4 . ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , or D-PBS as unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in A. Data are expressed as mean ± SD of three independent experiments. ( D ) PTX-sensitive secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 4 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. *: p value < 0.05, **: p value < 0.01 as compared with unstimulated cells. #: p -value < 0.05, ##: p value < 0.01 as compared with stimulated cells in isotype control group or vehicle control group.
    Figure Legend Snippet: Involvement of TLR2 and PTX-sensitive heterotrimeric G proteins in HP-NAP-induced secretion of IL-8 by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-dependent secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in . Data are expressed as mean ± SD of four independent experiments. ( B ) TLR2-dependent secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. ( C ) PTX-sensitive secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP and Pam 3 CSK 4 . ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , or D-PBS as unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in A. Data are expressed as mean ± SD of three independent experiments. ( D ) PTX-sensitive secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 4 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. *: p value < 0.05, **: p value < 0.01 as compared with unstimulated cells. #: p -value < 0.05, ##: p value < 0.01 as compared with stimulated cells in isotype control group or vehicle control group.

    Techniques Used:

    Specific interaction of HP-NAP with recombinant human TLR2 protein. ( A ) Binding of TLR2 to HP-NAP as determined by an ELISA-based solid-phase binding assay. The indicated amounts of recombinant human TLR2-10xHis and TLR4-10xHis proteins were incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 and TLR4 to HP-NAP was detected by anti-His tag antibody or its isotype control antibody using the ELISA-based solid-phase binding assay as described in Materials and Methods. The results are presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments. ( B ) Specific binding of TLR2 to HP-NAP as determined by competitive binding with Pam 3 CSK 4 . The indicated amounts of Pam 3 CSK 4 (Pam3) and BSA, as a negative control, were incubated with 0.625 μg of the recombinant human TLR2-10xHis protein at 4 °C overnight and then incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 to HP-NAP was detected by the ELISA-based solid-phase binding assay as described in A. The results were presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments.
    Figure Legend Snippet: Specific interaction of HP-NAP with recombinant human TLR2 protein. ( A ) Binding of TLR2 to HP-NAP as determined by an ELISA-based solid-phase binding assay. The indicated amounts of recombinant human TLR2-10xHis and TLR4-10xHis proteins were incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 and TLR4 to HP-NAP was detected by anti-His tag antibody or its isotype control antibody using the ELISA-based solid-phase binding assay as described in Materials and Methods. The results are presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments. ( B ) Specific binding of TLR2 to HP-NAP as determined by competitive binding with Pam 3 CSK 4 . The indicated amounts of Pam 3 CSK 4 (Pam3) and BSA, as a negative control, were incubated with 0.625 μg of the recombinant human TLR2-10xHis protein at 4 °C overnight and then incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 to HP-NAP was detected by the ELISA-based solid-phase binding assay as described in A. The results were presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments.

    Techniques Used: Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control

    Antibodies used in this study.
    Figure Legend Snippet: Antibodies used in this study.

    Techniques Used: Concentration Assay


    Structured Review

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    Abcam monoclonal mouse anti human tlr2
    Identification of macrophage receptors to cartilage fragments and functional blocking assay. ( A ) A predicated model of macrophage activation by cartilage fragments based on bioinformatic analysis. ( B ) Detection of MARCO, <t>TLR2</t> and ITGα5 in synovial tissues of OA patients. Representative images form sections of synovial tissues of 8 OA patients. Lower panels show section stained by rabbit IgG and mouse IgG2 as negative controls. Scale bars are indicated on each image. ( C ) Effects of functional blocking antibodies to MARCO, TLR2 and ITGα5 on the secretion of TNF-α. Macrophages were pretreated with 10 µg/ml for 30 min and then cultured with cartilage fragments. Results are presented as means ± standard errors of the means from 4 wells values for each treatment. Significant difference was determined by one-way ANOVA followed by Tukey’s multiple-comparison procedure. The results are representative of two independent experiments. Experiments were repeated twice for reproducibility of data.
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    1) Product Images from "Transcriptional profiling of murine macrophages stimulated with cartilage fragments revealed a strategy for treatment of progressive osteoarthritis"

    Article Title: Transcriptional profiling of murine macrophages stimulated with cartilage fragments revealed a strategy for treatment of progressive osteoarthritis

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-64515-1

    Identification of macrophage receptors to cartilage fragments and functional blocking assay. ( A ) A predicated model of macrophage activation by cartilage fragments based on bioinformatic analysis. ( B ) Detection of MARCO, TLR2 and ITGα5 in synovial tissues of OA patients. Representative images form sections of synovial tissues of 8 OA patients. Lower panels show section stained by rabbit IgG and mouse IgG2 as negative controls. Scale bars are indicated on each image. ( C ) Effects of functional blocking antibodies to MARCO, TLR2 and ITGα5 on the secretion of TNF-α. Macrophages were pretreated with 10 µg/ml for 30 min and then cultured with cartilage fragments. Results are presented as means ± standard errors of the means from 4 wells values for each treatment. Significant difference was determined by one-way ANOVA followed by Tukey’s multiple-comparison procedure. The results are representative of two independent experiments. Experiments were repeated twice for reproducibility of data.
    Figure Legend Snippet: Identification of macrophage receptors to cartilage fragments and functional blocking assay. ( A ) A predicated model of macrophage activation by cartilage fragments based on bioinformatic analysis. ( B ) Detection of MARCO, TLR2 and ITGα5 in synovial tissues of OA patients. Representative images form sections of synovial tissues of 8 OA patients. Lower panels show section stained by rabbit IgG and mouse IgG2 as negative controls. Scale bars are indicated on each image. ( C ) Effects of functional blocking antibodies to MARCO, TLR2 and ITGα5 on the secretion of TNF-α. Macrophages were pretreated with 10 µg/ml for 30 min and then cultured with cartilage fragments. Results are presented as means ± standard errors of the means from 4 wells values for each treatment. Significant difference was determined by one-way ANOVA followed by Tukey’s multiple-comparison procedure. The results are representative of two independent experiments. Experiments were repeated twice for reproducibility of data.

    Techniques Used: Functional Assay, Blocking Assay, Activation Assay, Staining, Cell Culture

    t2 5 monoclonal anti tlr2 ab  (Hycult Biotech)


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    Hycult Biotech t2 5 monoclonal anti tlr2 ab
    T2 5 Monoclonal Anti Tlr2 Ab, supplied by Hycult Biotech, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse monoclonal anti tlr2 r d systems cat  (R&D Systems)


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    R&D Systems mouse monoclonal anti tlr2 r d systems cat
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    Hycult Biotech t2 5 monoclonal anti tlr2 ab
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    Santa Cruz Biotechnology monoclonal mouse anti human tlr2 antibody
    <t>TLR2</t> and TLR4 were expressed on HK-2 cells and interacted with AGE-LDL. (A) Protein expression of TLR2 and TLR4 on the surface of HK-2 cells was detected by flow cytometry analysis. (B) Co-immunoprecipitation of AGE-LDL and TLR2/4 in HK-2 cells. HK-2 cells were treated with AGE-LDL, native LDL, or AGE-BSA, respectively, the interaction between AGE-LDL and TLR2 or TLR4 was analyzed by immunoprecipitation using anti-AGE antibody and detected by immunoblotting using anti-TLR2 or anti-TLR4 antibody, respectively. The protein level of TLR2 and TLR4 in HK-2 cell lysate was examined by western blot.
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    HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 <t>(TLR2/6;</t> 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.
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    ( A ) <t>TLR2</t> expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.
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    ( A ) <t>TLR2</t> expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.
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    Thermo Fisher functional graded monoclonal mouse anti human tlr2
    The effect of <t>TLR2-neutralizing</t> antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) <t>TLR2-independent</t> ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 10 6 cell/mL were pretreated with 10 μg/mL of <t>anti-TLR2,</t> anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in C and as mean ± SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.
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    Abnova mouse anti human tlr2 monoclonal igg
    The effect of <t>TLR2-neutralizing</t> antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) <t>TLR2-independent</t> ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 10 6 cell/mL were pretreated with 10 μg/mL of <t>anti-TLR2,</t> anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in C and as mean ± SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.
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    Abcam monoclonal mouse anti human tlr2
    Identification of macrophage receptors to cartilage fragments and functional blocking assay. ( A ) A predicated model of macrophage activation by cartilage fragments based on bioinformatic analysis. ( B ) Detection of MARCO, <t>TLR2</t> and ITGα5 in synovial tissues of OA patients. Representative images form sections of synovial tissues of 8 OA patients. Lower panels show section stained by rabbit IgG and mouse IgG2 as negative controls. Scale bars are indicated on each image. ( C ) Effects of functional blocking antibodies to MARCO, TLR2 and ITGα5 on the secretion of TNF-α. Macrophages were pretreated with 10 µg/ml for 30 min and then cultured with cartilage fragments. Results are presented as means ± standard errors of the means from 4 wells values for each treatment. Significant difference was determined by one-way ANOVA followed by Tukey’s multiple-comparison procedure. The results are representative of two independent experiments. Experiments were repeated twice for reproducibility of data.
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    Image Search Results


    TLR2 and TLR4 were expressed on HK-2 cells and interacted with AGE-LDL. (A) Protein expression of TLR2 and TLR4 on the surface of HK-2 cells was detected by flow cytometry analysis. (B) Co-immunoprecipitation of AGE-LDL and TLR2/4 in HK-2 cells. HK-2 cells were treated with AGE-LDL, native LDL, or AGE-BSA, respectively, the interaction between AGE-LDL and TLR2 or TLR4 was analyzed by immunoprecipitation using anti-AGE antibody and detected by immunoblotting using anti-TLR2 or anti-TLR4 antibody, respectively. The protein level of TLR2 and TLR4 in HK-2 cell lysate was examined by western blot.

    Journal: International Journal of Biological Sciences

    Article Title: AGE-LDL Activates Toll Like Receptor 4 Pathway and Promotes Inflammatory Cytokines Production in Renal Tubular Epithelial Cells

    doi: 10.7150/ijbs.5246

    Figure Lengend Snippet: TLR2 and TLR4 were expressed on HK-2 cells and interacted with AGE-LDL. (A) Protein expression of TLR2 and TLR4 on the surface of HK-2 cells was detected by flow cytometry analysis. (B) Co-immunoprecipitation of AGE-LDL and TLR2/4 in HK-2 cells. HK-2 cells were treated with AGE-LDL, native LDL, or AGE-BSA, respectively, the interaction between AGE-LDL and TLR2 or TLR4 was analyzed by immunoprecipitation using anti-AGE antibody and detected by immunoblotting using anti-TLR2 or anti-TLR4 antibody, respectively. The protein level of TLR2 and TLR4 in HK-2 cell lysate was examined by western blot.

    Article Snippet: After incubating with a monoclonal mouse anti-human TLR2 antibody or a polyclonal rabbit anti-human TLR4 antibody for 1 h at 4°C, HK-2 cells were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary IgG (Santa Cruz Biotechnology Inc. CA, USA) or FITC-conjugated goat anti-rabbit secondary (Santa Cruz Biotechnology Inc. CA, USA) for 45 min and analyzed by flow cytometry using FACS Calibur and CellQuest software (BD FACS Calibur System, Franklin Lakes, NJ, USA).

    Techniques: Expressing, Flow Cytometry, Immunoprecipitation, Western Blot

    AGE-LDL induced IL-6 and IL-8 production was mainly mediated by TLR4. (A) HK-2 cells were transfected with TLR2 siRNA, TLR4 siRNA or scramble siRNA, respectively. Western blot was performed to analyze the expression of endogenous TLR2/4 in HK-2 cells. GAPDH was used to verify equivalent loading. The effect of TLR2/4 siRNA on AGE-LDL-induced IL-6 and IL-8 expression was determined by real-time PCR (B, D) and ELISA (C, E). * P <0.05 versus untreated cells. # P <0.05 versus scramble siRNA transfected cells. Data are expressed as mean±SD of three independent experiments.

    Journal: International Journal of Biological Sciences

    Article Title: AGE-LDL Activates Toll Like Receptor 4 Pathway and Promotes Inflammatory Cytokines Production in Renal Tubular Epithelial Cells

    doi: 10.7150/ijbs.5246

    Figure Lengend Snippet: AGE-LDL induced IL-6 and IL-8 production was mainly mediated by TLR4. (A) HK-2 cells were transfected with TLR2 siRNA, TLR4 siRNA or scramble siRNA, respectively. Western blot was performed to analyze the expression of endogenous TLR2/4 in HK-2 cells. GAPDH was used to verify equivalent loading. The effect of TLR2/4 siRNA on AGE-LDL-induced IL-6 and IL-8 expression was determined by real-time PCR (B, D) and ELISA (C, E). * P <0.05 versus untreated cells. # P <0.05 versus scramble siRNA transfected cells. Data are expressed as mean±SD of three independent experiments.

    Article Snippet: After incubating with a monoclonal mouse anti-human TLR2 antibody or a polyclonal rabbit anti-human TLR4 antibody for 1 h at 4°C, HK-2 cells were then washed and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary IgG (Santa Cruz Biotechnology Inc. CA, USA) or FITC-conjugated goat anti-rabbit secondary (Santa Cruz Biotechnology Inc. CA, USA) for 45 min and analyzed by flow cytometry using FACS Calibur and CellQuest software (BD FACS Calibur System, Franklin Lakes, NJ, USA).

    Techniques: Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 (TLR2/6; 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.

    Journal: PLoS ONE

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    doi: 10.1371/journal.pone.0068701

    Figure Lengend Snippet: HASMCs were cultured for 24 h in the absence of presence of Pam 3 CSK 4 (TLR1/2; 1 µg/ml), FSL-1 (TLR2/6; 100 ng/ml), poly(I:C) (TLR3; 10 µg/ml), LPS (TLR4; 100 ng/ml), flagellin (TLR5; 1 µg/ml), R-837 (TLR7; 5 µg/ml), R-848 (TLR7/8; 5 µg/ml), CpG (TLR9; 1 µM), iE-DAP (NOD1; 10 µg/ml), MDP (NOD2; 10 µg/ml) and poly(I:C)/LyoVec (RIG-I/MDA-5; 1 µg/ml). Thereafter, the cell-free culture supernatants were recovered and analyzed for levels of ( A ) IL-6 (n = 14), ( B ) IL-8 (n = 14), ( C ) GM-CSF (n = 14), ( D ) eotaxin (n = 7), ( E ) RANTES (n = 7), and ( F ) TGF-β1 (n = 6) using ELISA. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.

    Article Snippet: The slides were incubated for 1 h at RT with mouse anti-human mAbs against TLR2 (TL2.3, eBioscience) and TLR3 (40C1285.6, AMS Biotechnology) or rabbit anti-human pAbs against TLR4, TLR7 and NOD1 (Abcam) at a dilution of 1∶10 and 1∶50.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

    ( A–E ) HASMCs were stained intracellularly with Abs against TLR2, TLR3, TLR4, TLR7 and NOD1 (open histograms) or appropriate isotype control (shaded histograms) and analyzed by flow cytometry. Data show one out of three independent experiments. ( F ) Data are presented as relative mean fluorescence intensity (rMFI = MFI Ab /MFI isotype control ) and depicted as mean ± SEM (n = 3). ( G ) Immunocytochemistry of HASMCs stained with Abs against TLR2, ( H ) TLR3 (diluted 1∶10), ( I ) TLR4, ( J ) TLR7, ( K ) NOD1 (diluted 1∶50), and ( L ) N-series negative control reagent (diluted 1∶10). Slides were visualized using 3,3′-diaminobenzidine (brown). All slides were counterstained with hematoxylin (blue) and analyzed by microscopy; magnification 200×.

    Journal: PLoS ONE

    Article Title: Innate Immune Receptors in Human Airway Smooth Muscle Cells: Activation by TLR1/2, TLR3, TLR4, TLR7 and NOD1 Agonists

    doi: 10.1371/journal.pone.0068701

    Figure Lengend Snippet: ( A–E ) HASMCs were stained intracellularly with Abs against TLR2, TLR3, TLR4, TLR7 and NOD1 (open histograms) or appropriate isotype control (shaded histograms) and analyzed by flow cytometry. Data show one out of three independent experiments. ( F ) Data are presented as relative mean fluorescence intensity (rMFI = MFI Ab /MFI isotype control ) and depicted as mean ± SEM (n = 3). ( G ) Immunocytochemistry of HASMCs stained with Abs against TLR2, ( H ) TLR3 (diluted 1∶10), ( I ) TLR4, ( J ) TLR7, ( K ) NOD1 (diluted 1∶50), and ( L ) N-series negative control reagent (diluted 1∶10). Slides were visualized using 3,3′-diaminobenzidine (brown). All slides were counterstained with hematoxylin (blue) and analyzed by microscopy; magnification 200×.

    Article Snippet: The slides were incubated for 1 h at RT with mouse anti-human mAbs against TLR2 (TL2.3, eBioscience) and TLR3 (40C1285.6, AMS Biotechnology) or rabbit anti-human pAbs against TLR4, TLR7 and NOD1 (Abcam) at a dilution of 1∶10 and 1∶50.

    Techniques: Staining, Flow Cytometry, Fluorescence, Immunocytochemistry, Negative Control, Microscopy

    ( A ) TLR2 expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.

    Journal: PLoS ONE

    Article Title: miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

    doi: 10.1371/journal.pone.0068296

    Figure Lengend Snippet: ( A ) TLR2 expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.

    Article Snippet: The cells were blocked for 1 h (BSA in PBS, pH 7.4) and then incubated with a mouse anti-human TLR2 mAb (Hycult Biotechnology) and a mouse anti-human CD133 mAb (Miltenyi Biotec) overnight at 4°C.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunofluorescence, Staining

    The effect of TLR2-neutralizing antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-independent ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in C and as mean ± SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.

    Journal: International Journal of Molecular Sciences

    Article Title: Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

    doi: 10.3390/ijms222111560

    Figure Lengend Snippet: The effect of TLR2-neutralizing antibodies on HP-NAP-induced ROS production by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-independent ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS, or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. ROS production was measured as DHE-derived fluorescence by flow cytometry as described in C. Representative histograms are shown in the left panel. Data in the right panel are expressed as the fold change as described in C and as mean ± SD of three independent experiments. ( B ) TLR2-independent ROS production in neutrophils induced by HP-NAP. Neutrophils at a density of 2 × 10 6 cell/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4 or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control for the indicated time. ROS production by neutrophils was determined by H 2 DCF-DA-derived derived fluorescence assay as described in A. Data from neutrophils stimulated with HP-NAP for 2.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in duplicate. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001 as compared with the unstimulated cells. ##: p value < 0.01, ###: p value < 0.001 as compared with HP-NAP-stimulated cells in the isotype control group. n.s.: non-significant.

    Article Snippet: For the blockade of TLR2 and TLR4, ATRA-induced differentiated HL-60 cells and neutrophils were pretreated with 10 μg/mL functional graded monoclonal mouse anti-human TLR2 (clone TL2.1) antibody (Cat. # 16-9922-82, eBioscience, San Diego, CA, USA), 10 μg/mL functional graded monoclonal mouse anti-human TLR4 (clone HTA125) antibody (Cat. # 16-9917-82, eBioscience), or 10 μg/mL functional graded mouse IgG 2a , kappa monoclonal (clone eBM2a) isotype antibody (Cat. # 16-4724-85, eBioscience) at 37 °C for 30 min.

    Techniques: Derivative Assay, Fluorescence, Flow Cytometry

    Involvement of TLR2 and PTX-sensitive heterotrimeric G proteins in HP-NAP-induced secretion of IL-8 by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-dependent secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in . Data are expressed as mean ± SD of four independent experiments. ( B ) TLR2-dependent secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. ( C ) PTX-sensitive secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP and Pam 3 CSK 4 . ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , or D-PBS as unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in A. Data are expressed as mean ± SD of three independent experiments. ( D ) PTX-sensitive secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 4 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. *: p value < 0.05, **: p value < 0.01 as compared with unstimulated cells. #: p -value < 0.05, ##: p value < 0.01 as compared with stimulated cells in isotype control group or vehicle control group.

    Journal: International Journal of Molecular Sciences

    Article Title: Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

    doi: 10.3390/ijms222111560

    Figure Lengend Snippet: Involvement of TLR2 and PTX-sensitive heterotrimeric G proteins in HP-NAP-induced secretion of IL-8 by ATRA-induced differentiated HL-60 cells and neutrophils. ( A ) TLR2-dependent secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in . Data are expressed as mean ± SD of four independent experiments. ( B ) TLR2-dependent secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 10 μg/mL of anti-TLR2, anti-TLR4, or IgG2a isotype antibodies at 37 °C for 30 min and followed by the stimulation with 1 μM HP-NAP, 1 μg/mL Pam 3 CSK 4 , 10 μg/mL LPS or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. ( C ) PTX-sensitive secretion of IL-8 from ATRA-induced differentiated HL-60 cells induced by HP-NAP and Pam 3 CSK 4 . ATRA-induced differentiated HL-60 cells at a density of 2 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 μM HP-NAP, 10 ng/mL Pam 3 CSK 4 , or D-PBS as unstimulated control at 37 °C for 16 h. The amount of IL-8 release was determined as described in A. Data are expressed as mean ± SD of three independent experiments. ( D ) PTX-sensitive secretion of IL-8 from neutrophils induced by HP-NAP. Neutrophils at a density of 5 × 10 6 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 4 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. The amount of IL-8 release was determined as described in A. Data from neutrophils stimulated with HP-NAP for 3 h are shown as the bar graph and expressed as mean ± SD of four independent experiments. *: p value < 0.05, **: p value < 0.01 as compared with unstimulated cells. #: p -value < 0.05, ##: p value < 0.01 as compared with stimulated cells in isotype control group or vehicle control group.

    Article Snippet: For the blockade of TLR2 and TLR4, ATRA-induced differentiated HL-60 cells and neutrophils were pretreated with 10 μg/mL functional graded monoclonal mouse anti-human TLR2 (clone TL2.1) antibody (Cat. # 16-9922-82, eBioscience, San Diego, CA, USA), 10 μg/mL functional graded monoclonal mouse anti-human TLR4 (clone HTA125) antibody (Cat. # 16-9917-82, eBioscience), or 10 μg/mL functional graded mouse IgG 2a , kappa monoclonal (clone eBM2a) isotype antibody (Cat. # 16-4724-85, eBioscience) at 37 °C for 30 min.

    Techniques:

    Specific interaction of HP-NAP with recombinant human TLR2 protein. ( A ) Binding of TLR2 to HP-NAP as determined by an ELISA-based solid-phase binding assay. The indicated amounts of recombinant human TLR2-10xHis and TLR4-10xHis proteins were incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 and TLR4 to HP-NAP was detected by anti-His tag antibody or its isotype control antibody using the ELISA-based solid-phase binding assay as described in Materials and Methods. The results are presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments. ( B ) Specific binding of TLR2 to HP-NAP as determined by competitive binding with Pam 3 CSK 4 . The indicated amounts of Pam 3 CSK 4 (Pam3) and BSA, as a negative control, were incubated with 0.625 μg of the recombinant human TLR2-10xHis protein at 4 °C overnight and then incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 to HP-NAP was detected by the ELISA-based solid-phase binding assay as described in A. The results were presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments.

    Journal: International Journal of Molecular Sciences

    Article Title: Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

    doi: 10.3390/ijms222111560

    Figure Lengend Snippet: Specific interaction of HP-NAP with recombinant human TLR2 protein. ( A ) Binding of TLR2 to HP-NAP as determined by an ELISA-based solid-phase binding assay. The indicated amounts of recombinant human TLR2-10xHis and TLR4-10xHis proteins were incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 and TLR4 to HP-NAP was detected by anti-His tag antibody or its isotype control antibody using the ELISA-based solid-phase binding assay as described in Materials and Methods. The results are presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments. ( B ) Specific binding of TLR2 to HP-NAP as determined by competitive binding with Pam 3 CSK 4 . The indicated amounts of Pam 3 CSK 4 (Pam3) and BSA, as a negative control, were incubated with 0.625 μg of the recombinant human TLR2-10xHis protein at 4 °C overnight and then incubated with 0.05 μg of HP-NAP coated on an ELISA plate. The binding of TLR2 to HP-NAP was detected by the ELISA-based solid-phase binding assay as described in A. The results were presented as absorbance at OD 450 nm . Data are expressed as mean ± SD of two independent experiments.

    Article Snippet: For the blockade of TLR2 and TLR4, ATRA-induced differentiated HL-60 cells and neutrophils were pretreated with 10 μg/mL functional graded monoclonal mouse anti-human TLR2 (clone TL2.1) antibody (Cat. # 16-9922-82, eBioscience, San Diego, CA, USA), 10 μg/mL functional graded monoclonal mouse anti-human TLR4 (clone HTA125) antibody (Cat. # 16-9917-82, eBioscience), or 10 μg/mL functional graded mouse IgG 2a , kappa monoclonal (clone eBM2a) isotype antibody (Cat. # 16-4724-85, eBioscience) at 37 °C for 30 min.

    Techniques: Recombinant, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control

    Antibodies used in this study.

    Journal: International Journal of Molecular Sciences

    Article Title: Helicobacter pylori Neutrophil-Activating Protein Directly Interacts with and Activates Toll-like Receptor 2 to Induce the Secretion of Interleukin-8 from Neutrophils and ATRA-Induced Differentiated HL-60 Cells

    doi: 10.3390/ijms222111560

    Figure Lengend Snippet: Antibodies used in this study.

    Article Snippet: For the blockade of TLR2 and TLR4, ATRA-induced differentiated HL-60 cells and neutrophils were pretreated with 10 μg/mL functional graded monoclonal mouse anti-human TLR2 (clone TL2.1) antibody (Cat. # 16-9922-82, eBioscience, San Diego, CA, USA), 10 μg/mL functional graded monoclonal mouse anti-human TLR4 (clone HTA125) antibody (Cat. # 16-9917-82, eBioscience), or 10 μg/mL functional graded mouse IgG 2a , kappa monoclonal (clone eBM2a) isotype antibody (Cat. # 16-4724-85, eBioscience) at 37 °C for 30 min.

    Techniques: Concentration Assay

    Identification of macrophage receptors to cartilage fragments and functional blocking assay. ( A ) A predicated model of macrophage activation by cartilage fragments based on bioinformatic analysis. ( B ) Detection of MARCO, TLR2 and ITGα5 in synovial tissues of OA patients. Representative images form sections of synovial tissues of 8 OA patients. Lower panels show section stained by rabbit IgG and mouse IgG2 as negative controls. Scale bars are indicated on each image. ( C ) Effects of functional blocking antibodies to MARCO, TLR2 and ITGα5 on the secretion of TNF-α. Macrophages were pretreated with 10 µg/ml for 30 min and then cultured with cartilage fragments. Results are presented as means ± standard errors of the means from 4 wells values for each treatment. Significant difference was determined by one-way ANOVA followed by Tukey’s multiple-comparison procedure. The results are representative of two independent experiments. Experiments were repeated twice for reproducibility of data.

    Journal: Scientific Reports

    Article Title: Transcriptional profiling of murine macrophages stimulated with cartilage fragments revealed a strategy for treatment of progressive osteoarthritis

    doi: 10.1038/s41598-020-64515-1

    Figure Lengend Snippet: Identification of macrophage receptors to cartilage fragments and functional blocking assay. ( A ) A predicated model of macrophage activation by cartilage fragments based on bioinformatic analysis. ( B ) Detection of MARCO, TLR2 and ITGα5 in synovial tissues of OA patients. Representative images form sections of synovial tissues of 8 OA patients. Lower panels show section stained by rabbit IgG and mouse IgG2 as negative controls. Scale bars are indicated on each image. ( C ) Effects of functional blocking antibodies to MARCO, TLR2 and ITGα5 on the secretion of TNF-α. Macrophages were pretreated with 10 µg/ml for 30 min and then cultured with cartilage fragments. Results are presented as means ± standard errors of the means from 4 wells values for each treatment. Significant difference was determined by one-way ANOVA followed by Tukey’s multiple-comparison procedure. The results are representative of two independent experiments. Experiments were repeated twice for reproducibility of data.

    Article Snippet: The slides were blocked with PBS-containing 1% bovine serum albumin plus 5% horse serum for 1 hour and incubated with primary antibodies including 1:50 monoclonal mouse anti-human TLR2 (Toll-like receptor 2) (Abcam, Cambridge, UK), 1:250 polyclonal rabbit anti-human MARCO (macrophage receptor with collagenous structure) (Atlas Antibodies, Stockholm, Sweden) and 1:100 monoclonal rabbit anti-human ITGα5 (integrin alpha 5) (Abcam) overnight at 4 °C.

    Techniques: Functional Assay, Blocking Assay, Activation Assay, Staining, Cell Culture