enzyme linked immunosorbent assay  (Sino Biological)


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    Name:
    Neuropilin 2 Protein Human Recombinant
    Description:
    A DNA sequence encoding the human NRP2 NP 003863 2 Met1 Tyr855 was expressed with the Fc region of human IgG1 at the C terminus
    Catalog Number:
    10695-H02H
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    Neuropilin-2 Protein Human, NP2 Protein Human, NPN2 Protein Human, PRO2714 Protein Human, VEGF165R2 Protein Human
    Host:
    HEK293 Cells
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    Structured Review

    Sino Biological enzyme linked immunosorbent assay
    A DNA sequence encoding the human NRP2 NP 003863 2 Met1 Tyr855 was expressed with the Fc region of human IgG1 at the C terminus
    https://www.bioz.com/result/enzyme linked immunosorbent assay/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    enzyme linked immunosorbent assay - by Bioz Stars, 2021-06
    86/100 stars

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    Related Articles

    Inhibition:

    Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus
    Article Snippet: The number of YFP positive cells was used to calculate % infection using media alone as 100%. .. Monoclonal antibody inhibition assay Virus (MOI:0.5) incubated with respective mAbs (2 μg ml−1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 104 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media. ..

    Incubation:

    Article Title: CD46 facilitates entry and dissemination of human cytomegalovirus
    Article Snippet: The number of YFP positive cells was used to calculate % infection using media alone as 100%. .. Monoclonal antibody inhibition assay Virus (MOI:0.5) incubated with respective mAbs (2 μg ml−1 , or otherwise noted) or soluble proteins Nrp2 (P) and CD46 (P) (Sino Biological (cat#: 10695-H08H and 12239-H08H) for 1 h 4 °C was used to infect ARPE-19, HTR-8/SVneo, or MRC5 cells (1 × 104 cell/well in a 96-well plate) for 2 h (37 °C) and then replaced with new media. ..

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  • 91
    Sino Biological recombinant rat cxcl16
    Acinar cell expression of <t>Cxcl16.</t> Cerulein (100 µg/kg) was injected into WT and Cxcl16 −/− mice every 1 h 8 times on 2 consecutive days as described in Fig. 2A . ( A ) Cxcl16 immunostaining of pancreatic frozen sections from WT mice (0 and 33 h) and Cxcl16 −/− mouse (33 h). ( B ) Dual immunofluorescence of Cxcl16 (Alexa Fluor 488) and amylase (Alexa Fluor 594), or Cxcl16 (Alexa Fluor 594) and F4/80 (Alexa Fluor 488). Most of Cxcl16-expressing on the cell surface was positive for cytoplasmic amylase expression. ( C ) Macrophage-depleted AP model (Left, protocol). Clodronate liposomes (100 mg/kg) were injected at 24 h before the first cerulein injection. F4/80 and Cxcl16 mRNA expression in the pancreas at 33 h, relative to those of control mice at 0 h, was evaluated at 33 h. *p
    Recombinant Rat Cxcl16, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 1 article reviews
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    93
    Sino Biological 4 1bb fusion protein treatment100
    Blockade of <t>4-1BB</t> signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.
    4 1bb Fusion Protein Treatment100, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/4 1bb fusion protein treatment100/product/Sino Biological
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    90
    Sino Biological anti c1 inh
    Proposed mechanism for Vag8 mediated complement evasion. (A) In the absence of Vag8, C1 and MBL/MASP complexes are formed on the bacterial surface which results in the activation of the CP and LP proteases. These proteases cleave C4 and C2 and give rise to the C3 convertase (C4bC2a). Any C1s, C1r, MASP-1 or MASP-2 not part of a bacterial surface bound complex remains inactive by association with <t>C1-inh</t> and hence free intact C4 and C2 is available for recruitment upon complement activation on bacteria. (B) In the presence of Vag8, either the secreted passenger or as part of an OMV, the interaction between C1s, C1r, MASP-1 or MASP-2 and C1-inh is interrupted as Vag8 hijacks this inhibitor. This results in the presence of active proteases in the bacterial environment which cleave and hence deplete the free C4 and C2. The lack of C4 and C2 to be cleaved and deposited on the bacterial membrane upon recognition of B pertussis by C1 and MBL complexes will lead to decreased complement deposition on the surface of B . pertussis and subsequent bacterial killing. Epithelial cells were adapted from Servier Medical Art, provided by Servier under a CC-BY 3.0 license (available at: http://www.servier.com/powerpoint-image-bank ).
    Anti C1 Inh, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Sino Biological recombinant human ror1 protein
    Antitumor activity of <t>ROR1-cFab</t> in ovarian cancer cells (A) Wound healing assay. Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 48 h and then subjected to wound healing assay. (B) Transwell migration assay. Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 24h and then subjected to Transwell migration assay. Data are shown as mean ± SD (n = 3, NS, not significant, * p
    Recombinant Human Ror1 Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant human ror1 protein/product/Sino Biological
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human ror1 protein - by Bioz Stars, 2021-06
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    Image Search Results


    Acinar cell expression of Cxcl16. Cerulein (100 µg/kg) was injected into WT and Cxcl16 −/− mice every 1 h 8 times on 2 consecutive days as described in Fig. 2A . ( A ) Cxcl16 immunostaining of pancreatic frozen sections from WT mice (0 and 33 h) and Cxcl16 −/− mouse (33 h). ( B ) Dual immunofluorescence of Cxcl16 (Alexa Fluor 488) and amylase (Alexa Fluor 594), or Cxcl16 (Alexa Fluor 594) and F4/80 (Alexa Fluor 488). Most of Cxcl16-expressing on the cell surface was positive for cytoplasmic amylase expression. ( C ) Macrophage-depleted AP model (Left, protocol). Clodronate liposomes (100 mg/kg) were injected at 24 h before the first cerulein injection. F4/80 and Cxcl16 mRNA expression in the pancreas at 33 h, relative to those of control mice at 0 h, was evaluated at 33 h. *p

    Journal: Scientific Reports

    Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

    doi: 10.1038/s41598-018-27200-y

    Figure Lengend Snippet: Acinar cell expression of Cxcl16. Cerulein (100 µg/kg) was injected into WT and Cxcl16 −/− mice every 1 h 8 times on 2 consecutive days as described in Fig. 2A . ( A ) Cxcl16 immunostaining of pancreatic frozen sections from WT mice (0 and 33 h) and Cxcl16 −/− mouse (33 h). ( B ) Dual immunofluorescence of Cxcl16 (Alexa Fluor 488) and amylase (Alexa Fluor 594), or Cxcl16 (Alexa Fluor 594) and F4/80 (Alexa Fluor 488). Most of Cxcl16-expressing on the cell surface was positive for cytoplasmic amylase expression. ( C ) Macrophage-depleted AP model (Left, protocol). Clodronate liposomes (100 mg/kg) were injected at 24 h before the first cerulein injection. F4/80 and Cxcl16 mRNA expression in the pancreas at 33 h, relative to those of control mice at 0 h, was evaluated at 33 h. *p

    Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R & D Systems).

    Techniques: Expressing, Injection, Mouse Assay, Immunostaining, Immunofluorescence

    Cxcl16 expression in acute necrotizing pancreatitis. ( A ) Protocols of acute necrotizing pancreatitis by repeated injection of cerulein. Cerulein (100 ug/kg) was injected every 1 h 8 times on 2 consecutive days into C57BL/6 mice. Four to five mice were sacrificed at the indicated time points. ( B ) Serum amylase level and ( C ) pathology score of the respective time-points. ( D ) H E sections in the pancreas of mice treated with acute necrotizing pancreatitis regimen at 0, 9, 24, and 33 h, respectively. The section at 33 h shows disrupted acinar architecture, vacuolization and necrosis of acinar cells, and inflammatory cell infiltration. ( E ) Pancreatic mRNA expressions of Cxcl16 , Tnfα , Il6 , and Cxcl2 were determined by quantitative RT-PCR analysis. *p

    Journal: Scientific Reports

    Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

    doi: 10.1038/s41598-018-27200-y

    Figure Lengend Snippet: Cxcl16 expression in acute necrotizing pancreatitis. ( A ) Protocols of acute necrotizing pancreatitis by repeated injection of cerulein. Cerulein (100 ug/kg) was injected every 1 h 8 times on 2 consecutive days into C57BL/6 mice. Four to five mice were sacrificed at the indicated time points. ( B ) Serum amylase level and ( C ) pathology score of the respective time-points. ( D ) H E sections in the pancreas of mice treated with acute necrotizing pancreatitis regimen at 0, 9, 24, and 33 h, respectively. The section at 33 h shows disrupted acinar architecture, vacuolization and necrosis of acinar cells, and inflammatory cell infiltration. ( E ) Pancreatic mRNA expressions of Cxcl16 , Tnfα , Il6 , and Cxcl2 were determined by quantitative RT-PCR analysis. *p

    Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R & D Systems).

    Techniques: Expressing, Injection, Mouse Assay, Quantitative RT-PCR

    Therapeutic effects of neutralizing Cxcl16 Ab in the necrotizing pancreatitis model. ( A ) Injection protocol of anti-Cxcl16 Ab in necrotizing pancreatitis model. ( B ) Serum amylase level, ( C ) representative H E sections, and pathology score of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( D ) Gr1 immunostained sections and neutrophil counts of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( E ) Pancreatic Ccl9 mRNA, pancreatic Ccl9 protein, and serum Ccl9 protein levels in control Ab or anti-Cxcl16 Ab-treated mice at 39 h. n = 5 in each group. *p

    Journal: Scientific Reports

    Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

    doi: 10.1038/s41598-018-27200-y

    Figure Lengend Snippet: Therapeutic effects of neutralizing Cxcl16 Ab in the necrotizing pancreatitis model. ( A ) Injection protocol of anti-Cxcl16 Ab in necrotizing pancreatitis model. ( B ) Serum amylase level, ( C ) representative H E sections, and pathology score of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( D ) Gr1 immunostained sections and neutrophil counts of control Ab or anti-Cxcl16 Ab-treated mice at 39 h. ( E ) Pancreatic Ccl9 mRNA, pancreatic Ccl9 protein, and serum Ccl9 protein levels in control Ab or anti-Cxcl16 Ab-treated mice at 39 h. n = 5 in each group. *p

    Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R & D Systems).

    Techniques: Injection, Mouse Assay

    Cxcl16 −/− mice were resistant to the development of necrotizing pancreatitis. Cerulein (100 ug/kg) was injected into Cxcl16 -intact ( WT ) and Cxcl16 -deficient ( Cxcl16 −/− ) mice (n = 5 in each group) every 1 h 8 times on 2 consecutive days as decribed in Fig. 2A. ( A ) Serum amylase levels and ( B ) pathology scores of WT and Cxcl16 −/− mice. ( C ) H E sections from WT and Cxcl16 −/− mice at 24 h and 33 h. ( D ) Immunostained (Gr1, F4/80, and CD3) sections from WT and Cxcl16 −/− mice at 33 h. ( E ) Semiquantitative assessment of infiltartion of neutrophils, macrophages, and T cells. The number of neutrophils, macrophages, and T cells were counted in 5 high powered fields in each pancreas sections of WT and Cxcl16 −/− mice at 33 h. ( F ) MPO levels determined by ELISA in the pancreatic lysates of WT and Cxcl16 −/− mice at 33 h. *p

    Journal: Scientific Reports

    Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

    doi: 10.1038/s41598-018-27200-y

    Figure Lengend Snippet: Cxcl16 −/− mice were resistant to the development of necrotizing pancreatitis. Cerulein (100 ug/kg) was injected into Cxcl16 -intact ( WT ) and Cxcl16 -deficient ( Cxcl16 −/− ) mice (n = 5 in each group) every 1 h 8 times on 2 consecutive days as decribed in Fig. 2A. ( A ) Serum amylase levels and ( B ) pathology scores of WT and Cxcl16 −/− mice. ( C ) H E sections from WT and Cxcl16 −/− mice at 24 h and 33 h. ( D ) Immunostained (Gr1, F4/80, and CD3) sections from WT and Cxcl16 −/− mice at 33 h. ( E ) Semiquantitative assessment of infiltartion of neutrophils, macrophages, and T cells. The number of neutrophils, macrophages, and T cells were counted in 5 high powered fields in each pancreas sections of WT and Cxcl16 −/− mice at 33 h. ( F ) MPO levels determined by ELISA in the pancreatic lysates of WT and Cxcl16 −/− mice at 33 h. *p

    Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R & D Systems).

    Techniques: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    Induction of Ccl9 by Cxcl16 in necrotizing acute pancreatitis. ( A ) Cytokine/chemokine array using pancreatic lysates from C57BL/6 mice and Cxcl16 −/− mice treated with necrotizing pancreatitis regimen as described in Fig. 2A . The relative expression of cytokines and chemokines is shown together with representative images. The data presented were obtained from WT and Cxcl16 −/− mice at 33 h, and WT on 0 h. ( B ) Ccl9 , Vcam-1 , and Ccl2 mRNA expression, relative to those of WT mice at 0 h, was assessed by qPCR in the pancreas of WT and Cxcl16 −/− mice at 33 h. ( C ) Ccl9 immunostaining of pancreatic frozen sections from WT mice at 0 h and 33 h, and Cxcl16 −/− mice at 33 h. *p

    Journal: Scientific Reports

    Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

    doi: 10.1038/s41598-018-27200-y

    Figure Lengend Snippet: Induction of Ccl9 by Cxcl16 in necrotizing acute pancreatitis. ( A ) Cytokine/chemokine array using pancreatic lysates from C57BL/6 mice and Cxcl16 −/− mice treated with necrotizing pancreatitis regimen as described in Fig. 2A . The relative expression of cytokines and chemokines is shown together with representative images. The data presented were obtained from WT and Cxcl16 −/− mice at 33 h, and WT on 0 h. ( B ) Ccl9 , Vcam-1 , and Ccl2 mRNA expression, relative to those of WT mice at 0 h, was assessed by qPCR in the pancreas of WT and Cxcl16 −/− mice at 33 h. ( C ) Ccl9 immunostaining of pancreatic frozen sections from WT mice at 0 h and 33 h, and Cxcl16 −/− mice at 33 h. *p

    Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R & D Systems).

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Immunostaining

    Serum cytokine/chemokine array in patients with AP. Serum levels of six chemokines, among 40 cytokines/chemokines investigated, were significantly altered in MAP and SAP patients. Serum levels of CCL21, CCL13, and CCL15 in MAP were significantly lower in patients than in controls. Serum levels of MIF were significantly lower in SAP patients than in MAP patients. Serum levels of CCL27 were significantly lower in SAP patients than in control patients. Serum levels of CXCL16 were significantly higher in SAP patients than in control patients. When Bonferroni method was adopted to correct multiple testing problem, only CXCL16 level was revealed to have a significant difference. MAP, mild acute pancreatitis; SAP, severe acute pancreatitis. Results were shown as mean ± SD.

    Journal: Scientific Reports

    Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

    doi: 10.1038/s41598-018-27200-y

    Figure Lengend Snippet: Serum cytokine/chemokine array in patients with AP. Serum levels of six chemokines, among 40 cytokines/chemokines investigated, were significantly altered in MAP and SAP patients. Serum levels of CCL21, CCL13, and CCL15 in MAP were significantly lower in patients than in controls. Serum levels of MIF were significantly lower in SAP patients than in MAP patients. Serum levels of CCL27 were significantly lower in SAP patients than in control patients. Serum levels of CXCL16 were significantly higher in SAP patients than in control patients. When Bonferroni method was adopted to correct multiple testing problem, only CXCL16 level was revealed to have a significant difference. MAP, mild acute pancreatitis; SAP, severe acute pancreatitis. Results were shown as mean ± SD.

    Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R & D Systems).

    Techniques:

    Expression of Ccl9 by pancreatic acinar cells. ( A ) Amylase secretion by the rat acinar cell line AR42J upon stimulation with various concentrations of cerulein. ( B ) Cxcl16 and Ccl9 mRNA expression by AR42J upon stimulation with cerulein. ( C ) Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −10 M) in combination with various concentrations of recombinant Cxcl16 protein (left). Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −7 M) in the presence of neutralizing anti-Cxcl16 Ab or control Ab (right). ( D ) Cxcl16 and Ccl9 mRNA expression by pancreatic acinar cells isolated from WT and Cxcl16 −/− mice upon stimulation with cerulein (10 −7 M). *p

    Journal: Scientific Reports

    Article Title: Chemokine CXCL16 mediates acinar cell necrosis in cerulein induced acute pancreatitis in mice

    doi: 10.1038/s41598-018-27200-y

    Figure Lengend Snippet: Expression of Ccl9 by pancreatic acinar cells. ( A ) Amylase secretion by the rat acinar cell line AR42J upon stimulation with various concentrations of cerulein. ( B ) Cxcl16 and Ccl9 mRNA expression by AR42J upon stimulation with cerulein. ( C ) Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −10 M) in combination with various concentrations of recombinant Cxcl16 protein (left). Ccl9 mRNA expression of AR42J upon stimulation with cerulein (10 −7 M) in the presence of neutralizing anti-Cxcl16 Ab or control Ab (right). ( D ) Cxcl16 and Ccl9 mRNA expression by pancreatic acinar cells isolated from WT and Cxcl16 −/− mice upon stimulation with cerulein (10 −7 M). *p

    Article Snippet: Cells were stimulated for 60 min with various concentrations of cerulein after 60-min preincubation with recombinant rat Cxcl16 (Sino Biological, Beijing, China), rat anti-Cxcl16 Ab , or rat normal IgG (R & D Systems).

    Techniques: Expressing, Recombinant, Isolation, Mouse Assay

    Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Blockade of 4-1BB signaling attenuates the secretion of pro-fibrotic mediators by MH-S cells. Transfected (Len-cont. and sh-4-1BB) MH-S cells were treated with or without NQDI 1 (10 µM), 4-1BBIg (10 µg/mL), or IgG1 (10 µg/mL) for 2 h, then exposed to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) Levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E,G) Western blots analysis of IκBα and phospho-IκBα. (F,H) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (I–K) The expressions of MMP12, MMP9, and monocyte chemoattractant protein-1 were detected by real-time polymerase chain reaction analysis ( n = 4). ELISA analysis of cytokines in the culture supernatants. (L) IL-1β, (M) IL-6, (N) tumor necrosis factor-α ( n = 4). Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant). The data were representative of three independent experiments.

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

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

    A model for alveolar macrophages (AMs) expressing 4-1BB in the regulation of pulmonary fibrosis through secreting pro-fibrotic mediators. The expression of 4-1BB increases in AMs in response to crystalline silica, which leads to elevated secretion of pro-inflammatory and pro-fibrotic cytokines, chemokines, and MMPs. These pro-fibrotic mediators promote pulmonary alveoli injury, the accumulation of monocytes, lymphocytes, and fibrocytes, and collagen deposition, resulting in pulmonary fibrosis.

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: A model for alveolar macrophages (AMs) expressing 4-1BB in the regulation of pulmonary fibrosis through secreting pro-fibrotic mediators. The expression of 4-1BB increases in AMs in response to crystalline silica, which leads to elevated secretion of pro-inflammatory and pro-fibrotic cytokines, chemokines, and MMPs. These pro-fibrotic mediators promote pulmonary alveoli injury, the accumulation of monocytes, lymphocytes, and fibrocytes, and collagen deposition, resulting in pulmonary fibrosis.

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Affinity Magnetic Separation, Expressing

    Expression of 4-1BB on CD4+ T cells. (A) Representative plots of flow cytometric analyses for 4-1BB on effector and naïve T cells. (B) The percentage of effector T cells expressing 4-1BB. (C,D) The frequency of effector and naïve T cells in CD4+ T cells from the lungs. (E) The percentage of naïve T cells expressing 4-1BB. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; ns, not significant).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Expression of 4-1BB on CD4+ T cells. (A) Representative plots of flow cytometric analyses for 4-1BB on effector and naïve T cells. (B) The percentage of effector T cells expressing 4-1BB. (C,D) The frequency of effector and naïve T cells in CD4+ T cells from the lungs. (E) The percentage of naïve T cells expressing 4-1BB. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; ns, not significant).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Expressing

    4-1BB expression on the mouse alveolar macrophage cell line, MH-S. MH-S cells were treated with crystalline silica (50 µg/cm 2 ) or saline for 12 h. (A,B) The percentage of MH-S cells expressing 4-1BB ( n = 4). (C) Western blot analysis of 4-1BB protein from whole cell lysates. (D) Quantification of the 4-1BB protein level relative to that of β-actin is shown ( n = 3). (E) Total RNA was isolated to analyze 4-1BB mRNA expression ( n = 4) relative to GAPDH. The data were representative of three independent experiments. Data were expressed as mean ± SEM (*** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: 4-1BB expression on the mouse alveolar macrophage cell line, MH-S. MH-S cells were treated with crystalline silica (50 µg/cm 2 ) or saline for 12 h. (A,B) The percentage of MH-S cells expressing 4-1BB ( n = 4). (C) Western blot analysis of 4-1BB protein from whole cell lysates. (D) Quantification of the 4-1BB protein level relative to that of β-actin is shown ( n = 3). (E) Total RNA was isolated to analyze 4-1BB mRNA expression ( n = 4) relative to GAPDH. The data were representative of three independent experiments. Data were expressed as mean ± SEM (*** p ≤ 0.001).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Expressing, Western Blot, Isolation

    Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Activation of 4-1BB signaling promotes the secretion of pro-fibrotic mediators by MH-S cells. MH-S cells treated with or without agonist 4-1BB mAb (10 µg/mL) or IgG (10 µg/mL) for 2 h prior to exposure to crystalline silica (50 µg/cm 2 ) for 12 h. (A) Western blots analysis of ASK-1 and downstream mitogen-activated protein kinase proteins (p38 and JNK/stress activated protein kinase) and their phosphorylated forms. (B–D) The levels of phospho-ASK1, phospho-p38, and phospho-JNK were normalized to those of β-actin ( n = 3). (E) Western blots analysis of IκBα and phospho-IκBα. (F) The level of phospho-IκBα was normalized to those of β-actin ( n = 3). (G–I) Real-time polymerase chain reaction analysis of MMP12, MMP9, and monocyte chemoattractant protein-1 mRNA expression ( n = 4). (J–L) ELISA analysis was used to quantify the secretion of IL-1β, IL-6 and tumor necrosis factor-α ( n = 4). The results were representative of three independent experiments. Results were graphed as the mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

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

    The secretion of pro-fibrotic mediators is reduced in the lungs from crystalline silica (CS)-injured mice, upon inhibition of 4-1BB signaling. C57BL/6 mice were administered a CS suspension or saline, respectively; 4-1BBIg or isotype control (IgG1) were injected intraperitoneally (i.p.; n = 3–4). (A–D) Quantification of MMP9 and MMP12 protein levels by western blot, which were normalized to those of β-actin in lungs. Shown as bar graph. (E–H) ELISA analysis of cytokines in lung tissues. (E) IL-1β, (F) IL-6, (G) tumor necrosis factor-α, (H) monocyte chemoattractant protein-1. Experiments were performed three times. C57BL/6 mice were administered a CS suspension or saline, respectively; NQDI 1 or isotype control were injected i.p. ( n = 10). (I) Immunohistochemical staining of paraffin-embedded lung tissue sections at 7 and 56 days showed CD68, MMP9, and MMP12 expression. Nuclei were stained by hematoxylin (blue). (J–L) Identification of MMP9 and MMP12 protein levels in mouse lung tissues at 7 and 56 days by western blot. The levels of MMP9 and MMP12 were normalized to those of β-actin. (M) Representative images for the immunohistochemical staining of collagen I in paraffin-embedded lung tissue sections 56 days after CS instillation. Nuclei were stained by hematoxylin (blue). (I,M) Scale bar, 50 µm. Experiments were performed three times. Data are shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: The secretion of pro-fibrotic mediators is reduced in the lungs from crystalline silica (CS)-injured mice, upon inhibition of 4-1BB signaling. C57BL/6 mice were administered a CS suspension or saline, respectively; 4-1BBIg or isotype control (IgG1) were injected intraperitoneally (i.p.; n = 3–4). (A–D) Quantification of MMP9 and MMP12 protein levels by western blot, which were normalized to those of β-actin in lungs. Shown as bar graph. (E–H) ELISA analysis of cytokines in lung tissues. (E) IL-1β, (F) IL-6, (G) tumor necrosis factor-α, (H) monocyte chemoattractant protein-1. Experiments were performed three times. C57BL/6 mice were administered a CS suspension or saline, respectively; NQDI 1 or isotype control were injected i.p. ( n = 10). (I) Immunohistochemical staining of paraffin-embedded lung tissue sections at 7 and 56 days showed CD68, MMP9, and MMP12 expression. Nuclei were stained by hematoxylin (blue). (J–L) Identification of MMP9 and MMP12 protein levels in mouse lung tissues at 7 and 56 days by western blot. The levels of MMP9 and MMP12 were normalized to those of β-actin. (M) Representative images for the immunohistochemical staining of collagen I in paraffin-embedded lung tissue sections 56 days after CS instillation. Nuclei were stained by hematoxylin (blue). (I,M) Scale bar, 50 µm. Experiments were performed three times. Data are shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Mouse Assay, Inhibition, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Expressing

    Expression of 4-1BB (CD137) and 4-1BBL (CD137L) on pulmonary macrophages. After 7-days exposure to crystalline silica, mice were sacrificed. The lungs were prepared as single-cell suspensions for flow cytometric analyses ( n = 3–4). (A) Representative plots of flow cytometric analyses for 4-1BB and 4-1BBL on alveolar macrophages (AMs) and interstitial macrophages (IMs). (B,C) The percentage of AMs expressing 4-1BB and 4-1BBL. (D,E) The frequency of AMs and IMs in CD45+ cells from the lungs. (F,G) The percentage of IMs expressing 4-1BB and 4-1BBL. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Journal: Frontiers in Immunology

    Article Title: 4-1BB Signaling Promotes Alveolar Macrophages-Mediated Pro-Fibrotic Responses and Crystalline Silica-Induced Pulmonary Fibrosis in Mice

    doi: 10.3389/fimmu.2018.01848

    Figure Lengend Snippet: Expression of 4-1BB (CD137) and 4-1BBL (CD137L) on pulmonary macrophages. After 7-days exposure to crystalline silica, mice were sacrificed. The lungs were prepared as single-cell suspensions for flow cytometric analyses ( n = 3–4). (A) Representative plots of flow cytometric analyses for 4-1BB and 4-1BBL on alveolar macrophages (AMs) and interstitial macrophages (IMs). (B,C) The percentage of AMs expressing 4-1BB and 4-1BBL. (D,E) The frequency of AMs and IMs in CD45+ cells from the lungs. (F,G) The percentage of IMs expressing 4-1BB and 4-1BBL. The experiments were performed twice with similar results. Data were shown as mean ± SEM (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant).

    Article Snippet: For 4-1BB Fusion Protein Treatment100 or 50 µg of 4-1BB fusion protein (4-1BBIg; cat: 50811-M02H, Sino Biological Inc., Beijing, China) per mouse or 100 µg isotype IgG (human IgG1; Sino Biological Inc.) were injected intraperitoneally into mice, on days 1 and 4 after CS administration (3–4 mice per group).

    Techniques: Expressing, Mouse Assay, Affinity Magnetic Separation

    Proposed mechanism for Vag8 mediated complement evasion. (A) In the absence of Vag8, C1 and MBL/MASP complexes are formed on the bacterial surface which results in the activation of the CP and LP proteases. These proteases cleave C4 and C2 and give rise to the C3 convertase (C4bC2a). Any C1s, C1r, MASP-1 or MASP-2 not part of a bacterial surface bound complex remains inactive by association with C1-inh and hence free intact C4 and C2 is available for recruitment upon complement activation on bacteria. (B) In the presence of Vag8, either the secreted passenger or as part of an OMV, the interaction between C1s, C1r, MASP-1 or MASP-2 and C1-inh is interrupted as Vag8 hijacks this inhibitor. This results in the presence of active proteases in the bacterial environment which cleave and hence deplete the free C4 and C2. The lack of C4 and C2 to be cleaved and deposited on the bacterial membrane upon recognition of B pertussis by C1 and MBL complexes will lead to decreased complement deposition on the surface of B . pertussis and subsequent bacterial killing. Epithelial cells were adapted from Servier Medical Art, provided by Servier under a CC-BY 3.0 license (available at: http://www.servier.com/powerpoint-image-bank ).

    Journal: PLoS Pathogens

    Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

    doi: 10.1371/journal.ppat.1006531

    Figure Lengend Snippet: Proposed mechanism for Vag8 mediated complement evasion. (A) In the absence of Vag8, C1 and MBL/MASP complexes are formed on the bacterial surface which results in the activation of the CP and LP proteases. These proteases cleave C4 and C2 and give rise to the C3 convertase (C4bC2a). Any C1s, C1r, MASP-1 or MASP-2 not part of a bacterial surface bound complex remains inactive by association with C1-inh and hence free intact C4 and C2 is available for recruitment upon complement activation on bacteria. (B) In the presence of Vag8, either the secreted passenger or as part of an OMV, the interaction between C1s, C1r, MASP-1 or MASP-2 and C1-inh is interrupted as Vag8 hijacks this inhibitor. This results in the presence of active proteases in the bacterial environment which cleave and hence deplete the free C4 and C2. The lack of C4 and C2 to be cleaved and deposited on the bacterial membrane upon recognition of B pertussis by C1 and MBL complexes will lead to decreased complement deposition on the surface of B . pertussis and subsequent bacterial killing. Epithelial cells were adapted from Servier Medical Art, provided by Servier under a CC-BY 3.0 license (available at: http://www.servier.com/powerpoint-image-bank ).

    Article Snippet: The reaction was stopped by addition of 2x SB DTT and the effect was assessed by immunoblotting as described below by using anti-C1-inh (Sino Biologicals).

    Techniques: Activation Assay

    Vag8 mediates degradation of C4 and C2. (A) Purified C1s and C2 were incubated alone, or with C1-inh, in the presence of Vag8 or Prn and visualized using Instant Blue. Increased C2 cleavage is shown in the presence of Vag8. B . pertussis B1917 was opsonized with 1.25% NHS with or without Vag8 or Prn. Supernatant was analysed by immunoblot and shows cleavage of (B) C4, (C) C2, but not (D) C3 in the presence of Vag8 compared to control situation. Incubation of 1.25% NHS alone with or without Vag8 or Prn shows increased cleavage of (E) C4 and (F) C2 in the presence of Vag8. All figures are representative for three separate experiments.

    Journal: PLoS Pathogens

    Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

    doi: 10.1371/journal.ppat.1006531

    Figure Lengend Snippet: Vag8 mediates degradation of C4 and C2. (A) Purified C1s and C2 were incubated alone, or with C1-inh, in the presence of Vag8 or Prn and visualized using Instant Blue. Increased C2 cleavage is shown in the presence of Vag8. B . pertussis B1917 was opsonized with 1.25% NHS with or without Vag8 or Prn. Supernatant was analysed by immunoblot and shows cleavage of (B) C4, (C) C2, but not (D) C3 in the presence of Vag8 compared to control situation. Incubation of 1.25% NHS alone with or without Vag8 or Prn shows increased cleavage of (E) C4 and (F) C2 in the presence of Vag8. All figures are representative for three separate experiments.

    Article Snippet: The reaction was stopped by addition of 2x SB DTT and the effect was assessed by immunoblotting as described below by using anti-C1-inh (Sino Biologicals).

    Techniques: Purification, Incubation

    Vag8 binds C1-inh and is involved in serum resistance. (A) Vag8 binds to C1-inh in a dose dependent manner. No binding was observed to the serpins alpha-1-antichymotrypsin and alpha-2-antiplasmin or the complement components C1, C2, C3 and C4 by ELISA. (B) Vag8 forms a stable complex with C1-inh in fluid phase as shown by making use of the gel filtration chromatography method. (C) The successful construction of the B . pertussis B1917ΔVag8 mutant strain was confirmed by immunoblot. No Vag8 could be detected in the pellet or the bacterial supernatant or OMV’s of B1917ΔVag8. In addition, the B . pertussis wild type strain B1917 expressed both the full length Vag8 as well as the passenger domain in the supernatant. (D) Using ImageJ, the intensity of the Vag8 bands were semi quantified relative to known concentrations of recombinant Vag8. We show that 10 7 bacteria of the B1917 parental strain contain 10 μg/ml Vag8 and 10 9 bacteria secrete 5–10 μg/ml of full length and 1 μg/ml of passenger Vag8. Moreover, 10 μg/ml OMV contains 5 μg/ml of Vag8. (E) The B1917ΔVag8 mutant strain shows increased sensitivity to serum-mediated killing compared to the B1917 parent strain. Data shown in Fig 1A, 1D and 1E represent the mean ± SEM of three separate experiments while Fig 1B and 1C are representative of three separate experiments.

    Journal: PLoS Pathogens

    Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

    doi: 10.1371/journal.ppat.1006531

    Figure Lengend Snippet: Vag8 binds C1-inh and is involved in serum resistance. (A) Vag8 binds to C1-inh in a dose dependent manner. No binding was observed to the serpins alpha-1-antichymotrypsin and alpha-2-antiplasmin or the complement components C1, C2, C3 and C4 by ELISA. (B) Vag8 forms a stable complex with C1-inh in fluid phase as shown by making use of the gel filtration chromatography method. (C) The successful construction of the B . pertussis B1917ΔVag8 mutant strain was confirmed by immunoblot. No Vag8 could be detected in the pellet or the bacterial supernatant or OMV’s of B1917ΔVag8. In addition, the B . pertussis wild type strain B1917 expressed both the full length Vag8 as well as the passenger domain in the supernatant. (D) Using ImageJ, the intensity of the Vag8 bands were semi quantified relative to known concentrations of recombinant Vag8. We show that 10 7 bacteria of the B1917 parental strain contain 10 μg/ml Vag8 and 10 9 bacteria secrete 5–10 μg/ml of full length and 1 μg/ml of passenger Vag8. Moreover, 10 μg/ml OMV contains 5 μg/ml of Vag8. (E) The B1917ΔVag8 mutant strain shows increased sensitivity to serum-mediated killing compared to the B1917 parent strain. Data shown in Fig 1A, 1D and 1E represent the mean ± SEM of three separate experiments while Fig 1B and 1C are representative of three separate experiments.

    Article Snippet: The reaction was stopped by addition of 2x SB DTT and the effect was assessed by immunoblotting as described below by using anti-C1-inh (Sino Biologicals).

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Filtration, Chromatography, Mutagenesis, Recombinant

    Vag8 interferes with the binding of C1s, C1r and MASP-2 with C1-inh. (A) B . pertussis strain B1917 was opsonized with 1.25% NHS with or without Vag8 or Prn. Supernatant was analysed using anti C1-inh. The presence of Vag8 results in a decreased signal of the ~150 kDa band. (B) In a purified system consisting of C1q, C1s or C1r with C1-inh in the presence or absence of Vag8 or Prn, we show, using anti-C1-inh, that C1s and C1r, but not C1q, bind to C1-inh and that this binding is inhibited by the addition of Vag8 but not Prn. (C) C1r was detected using anti-C1r. The presence of C1r bound to C1 and C1-inh is inhibited in the presence of Vag8 but not Prn. Additionally, we show the presence of active C1r in the presence of Vag8. (D) Purified MASP-2 was incubated with C1-inh in the presence of Vag8 or Prn and analyzed using anti-C1-inh. We show inhibition of MASP-2 binding to C1-inh in the presence of Vag8, but not Prn. Immunoblots are representative of three separate experiments.

    Journal: PLoS Pathogens

    Article Title: Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

    doi: 10.1371/journal.ppat.1006531

    Figure Lengend Snippet: Vag8 interferes with the binding of C1s, C1r and MASP-2 with C1-inh. (A) B . pertussis strain B1917 was opsonized with 1.25% NHS with or without Vag8 or Prn. Supernatant was analysed using anti C1-inh. The presence of Vag8 results in a decreased signal of the ~150 kDa band. (B) In a purified system consisting of C1q, C1s or C1r with C1-inh in the presence or absence of Vag8 or Prn, we show, using anti-C1-inh, that C1s and C1r, but not C1q, bind to C1-inh and that this binding is inhibited by the addition of Vag8 but not Prn. (C) C1r was detected using anti-C1r. The presence of C1r bound to C1 and C1-inh is inhibited in the presence of Vag8 but not Prn. Additionally, we show the presence of active C1r in the presence of Vag8. (D) Purified MASP-2 was incubated with C1-inh in the presence of Vag8 or Prn and analyzed using anti-C1-inh. We show inhibition of MASP-2 binding to C1-inh in the presence of Vag8, but not Prn. Immunoblots are representative of three separate experiments.

    Article Snippet: The reaction was stopped by addition of 2x SB DTT and the effect was assessed by immunoblotting as described below by using anti-C1-inh (Sino Biologicals).

    Techniques: Binding Assay, Purification, Incubation, Inhibition, Western Blot

    Antitumor activity of ROR1-cFab in ovarian cancer cells (A) Wound healing assay. Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 48 h and then subjected to wound healing assay. (B) Transwell migration assay. Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 24h and then subjected to Transwell migration assay. Data are shown as mean ± SD (n = 3, NS, not significant, * p

    Journal: Oncotarget

    Article Title: Antitumor activity of a newly developed monoclonal antibody against ROR1 in ovarian cancer cells

    doi: 10.18632/oncotarget.21618

    Figure Lengend Snippet: Antitumor activity of ROR1-cFab in ovarian cancer cells (A) Wound healing assay. Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 48 h and then subjected to wound healing assay. (B) Transwell migration assay. Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 24h and then subjected to Transwell migration assay. Data are shown as mean ± SD (n = 3, NS, not significant, * p

    Article Snippet: Following this, 50 μL supernatant from each well with replicating fused cells was collected and added into a fresh 96-well plate that was pre-coated with 50 ng recombinant human ROR1 protein (Sino Biological Inc., Beijing, China).

    Techniques: Activity Assay, Wound Healing Assay, Transwell Migration Assay

    Confirmation of ROR1-cFab specificity and selectivity (A) ELISA. A 96-well plate was pre-coated with recombinant human ROR1 protein at a concentration of 50 ng/well. Serial dilutions of the ROR1-cFab were used as the first antibody for ELISA and the HRP-conjugated goat anti-human antibody (Fab specific) was used as the secondary antibody. Commercial anti-ROR1 antibody was used as a positive control (Ctrl). The absorbance was read at 450 nm after color development. (B) Surface Plasmon resonance (SPR) analysis. The ROR1 protein was diluted to 30 μg/mL in dilution buffer and then reacted in a running buffer containing serial concentrations of ROR1-cFab. Results were analyzed by the Biacore X100 software. The experiments were in triplicate and repeated at least twice. Data are shown as mean ± SD (n = 2, NS, not significant, * p

    Journal: Oncotarget

    Article Title: Antitumor activity of a newly developed monoclonal antibody against ROR1 in ovarian cancer cells

    doi: 10.18632/oncotarget.21618

    Figure Lengend Snippet: Confirmation of ROR1-cFab specificity and selectivity (A) ELISA. A 96-well plate was pre-coated with recombinant human ROR1 protein at a concentration of 50 ng/well. Serial dilutions of the ROR1-cFab were used as the first antibody for ELISA and the HRP-conjugated goat anti-human antibody (Fab specific) was used as the secondary antibody. Commercial anti-ROR1 antibody was used as a positive control (Ctrl). The absorbance was read at 450 nm after color development. (B) Surface Plasmon resonance (SPR) analysis. The ROR1 protein was diluted to 30 μg/mL in dilution buffer and then reacted in a running buffer containing serial concentrations of ROR1-cFab. Results were analyzed by the Biacore X100 software. The experiments were in triplicate and repeated at least twice. Data are shown as mean ± SD (n = 2, NS, not significant, * p

    Article Snippet: Following this, 50 μL supernatant from each well with replicating fused cells was collected and added into a fresh 96-well plate that was pre-coated with 50 ng recombinant human ROR1 protein (Sino Biological Inc., Beijing, China).

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Positive Control, SPR Assay, Software

    Assessment of the ROR1-cFab specificity in ovarian cancer cells (A) Western blot. It detected the level of ROR1 expression in ovarian cancer cells. Lane 1: supernatant of A2780 cell lysate; Lane 2: supernatant of Iose386 cell lysate. (B) Flow cytometry. Ovarian cancer A2780 and Iose386 cells were treated with or without ROR1-cFab for 1h and then incubated with a goat anti-human IgG (Fab specific)-FITC antibody for 1 h in the dark. The cells were then subjected to flow cytometry analysis of fluorescence intensity. (C) Immunofluorescence staining of ROR1. Ovarian cancer cells (A2780 and Iose386) were first grown and immunostained with ROR1-cFab. The experiments were in triplicate and repeated at least once.

    Journal: Oncotarget

    Article Title: Antitumor activity of a newly developed monoclonal antibody against ROR1 in ovarian cancer cells

    doi: 10.18632/oncotarget.21618

    Figure Lengend Snippet: Assessment of the ROR1-cFab specificity in ovarian cancer cells (A) Western blot. It detected the level of ROR1 expression in ovarian cancer cells. Lane 1: supernatant of A2780 cell lysate; Lane 2: supernatant of Iose386 cell lysate. (B) Flow cytometry. Ovarian cancer A2780 and Iose386 cells were treated with or without ROR1-cFab for 1h and then incubated with a goat anti-human IgG (Fab specific)-FITC antibody for 1 h in the dark. The cells were then subjected to flow cytometry analysis of fluorescence intensity. (C) Immunofluorescence staining of ROR1. Ovarian cancer cells (A2780 and Iose386) were first grown and immunostained with ROR1-cFab. The experiments were in triplicate and repeated at least once.

    Article Snippet: Following this, 50 μL supernatant from each well with replicating fused cells was collected and added into a fresh 96-well plate that was pre-coated with 50 ng recombinant human ROR1 protein (Sino Biological Inc., Beijing, China).

    Techniques: Western Blot, Expressing, Flow Cytometry, Incubation, Fluorescence, Immunofluorescence, Staining

    Cell viability CCK8 assay (A) A2780 cells and (B) Iose386 cells were grown and treated with different doses of ROR1-cFab for up to 72 h and then subjected to CCK8 assay.

    Journal: Oncotarget

    Article Title: Antitumor activity of a newly developed monoclonal antibody against ROR1 in ovarian cancer cells

    doi: 10.18632/oncotarget.21618

    Figure Lengend Snippet: Cell viability CCK8 assay (A) A2780 cells and (B) Iose386 cells were grown and treated with different doses of ROR1-cFab for up to 72 h and then subjected to CCK8 assay.

    Article Snippet: Following this, 50 μL supernatant from each well with replicating fused cells was collected and added into a fresh 96-well plate that was pre-coated with 50 ng recombinant human ROR1 protein (Sino Biological Inc., Beijing, China).

    Techniques: CCK-8 Assay

    Flow cytometric apoptosis assay Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 24h and then subjected to flow cytometric apoptosis assay. As a ROR1-negative cell line, Iose386 showed no difference in these assays. Data are shown as mean ± SD (n = 3, NS, not significant, * p

    Journal: Oncotarget

    Article Title: Antitumor activity of a newly developed monoclonal antibody against ROR1 in ovarian cancer cells

    doi: 10.18632/oncotarget.21618

    Figure Lengend Snippet: Flow cytometric apoptosis assay Tumor cells were grown and treated with 40 μg/mL of ROR1-cFab for up to 24h and then subjected to flow cytometric apoptosis assay. As a ROR1-negative cell line, Iose386 showed no difference in these assays. Data are shown as mean ± SD (n = 3, NS, not significant, * p

    Article Snippet: Following this, 50 μL supernatant from each well with replicating fused cells was collected and added into a fresh 96-well plate that was pre-coated with 50 ng recombinant human ROR1 protein (Sino Biological Inc., Beijing, China).

    Techniques: Apoptosis Assay

    Screening and identification of positive fusion cell clones and generation of chimeric monoclonal antibody ROR1-cFab (A) ELISA. ELISA was performed to screen 31 candidate fusion cell clones after three rounds of sub-clone screening. NC, a negative control. (B) Agarose gels of PCR amplification of the light chain. Lane 1, V L ; Lane 2, C L ; Lane 3, V L combined with C L ; M, a DNA maker. (C) Agarose gels of PCR amplification of the heavy chain. Lane 1, V H ; Lane 2, C H 1; Lane 3, V H combined with C H 1; M, a DNA maker. (D) Agarose gels of PCR amplification of pETDuet-ROR1-cFab. Lane 1, the plasmid of pETDuet without restriction endonuclease digestion; Lane 2, the plasmid of pETDuet-L; Lane 3, NcoI/HindIII were used for double digesting the pETDuet-L plasmid; Lane 4, the plasmid of pETDuet-L-H (pETDuet-ROR1-cFab); Lane 5, NdeI/kpnI were used for double digestion of the pETDuet-ROR1-cFab plasmid; M, a DNA marker. (E) Agarose gels of PCR amplification of the light and heavy chain of pETDuet-ROR1-cFab. Lane 1, L; Lane 2, Fd; M, a DNA maker. (F) Coomassie blue staining of a SDS-PAGE gel and (G) Western blot. Detection of expression of the Fab fragment in E. coli. Lane 1, supernatant of sonicated lysate of untransfected E. coli BL21, a negative control; Lane 2, sediment of sonicated lysate of untransfected E. coli BL21, a negative control; Lane 3, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli; Lane 4, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli; Lane 5, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; Lane 6, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; M, a protein marker. (H) Coomassie blue staining and (I) Western blot. Detection of the purification efficiency of the Fab fragments. Lane 1, the Fab fragments was purified by the Protein L affinity column; lane 2, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; lane 3, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; lane 4, the flow through the Protein L affinity column; M, a protein marker.

    Journal: Oncotarget

    Article Title: Antitumor activity of a newly developed monoclonal antibody against ROR1 in ovarian cancer cells

    doi: 10.18632/oncotarget.21618

    Figure Lengend Snippet: Screening and identification of positive fusion cell clones and generation of chimeric monoclonal antibody ROR1-cFab (A) ELISA. ELISA was performed to screen 31 candidate fusion cell clones after three rounds of sub-clone screening. NC, a negative control. (B) Agarose gels of PCR amplification of the light chain. Lane 1, V L ; Lane 2, C L ; Lane 3, V L combined with C L ; M, a DNA maker. (C) Agarose gels of PCR amplification of the heavy chain. Lane 1, V H ; Lane 2, C H 1; Lane 3, V H combined with C H 1; M, a DNA maker. (D) Agarose gels of PCR amplification of pETDuet-ROR1-cFab. Lane 1, the plasmid of pETDuet without restriction endonuclease digestion; Lane 2, the plasmid of pETDuet-L; Lane 3, NcoI/HindIII were used for double digesting the pETDuet-L plasmid; Lane 4, the plasmid of pETDuet-L-H (pETDuet-ROR1-cFab); Lane 5, NdeI/kpnI were used for double digestion of the pETDuet-ROR1-cFab plasmid; M, a DNA marker. (E) Agarose gels of PCR amplification of the light and heavy chain of pETDuet-ROR1-cFab. Lane 1, L; Lane 2, Fd; M, a DNA maker. (F) Coomassie blue staining of a SDS-PAGE gel and (G) Western blot. Detection of expression of the Fab fragment in E. coli. Lane 1, supernatant of sonicated lysate of untransfected E. coli BL21, a negative control; Lane 2, sediment of sonicated lysate of untransfected E. coli BL21, a negative control; Lane 3, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli; Lane 4, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli; Lane 5, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; Lane 6, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; M, a protein marker. (H) Coomassie blue staining and (I) Western blot. Detection of the purification efficiency of the Fab fragments. Lane 1, the Fab fragments was purified by the Protein L affinity column; lane 2, sediment of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; lane 3, supernatant of sonicated lysate of pETDuet-ROR1-cFab-transfected E. coli induced by IPTG overnight; lane 4, the flow through the Protein L affinity column; M, a protein marker.

    Article Snippet: Following this, 50 μL supernatant from each well with replicating fused cells was collected and added into a fresh 96-well plate that was pre-coated with 50 ng recombinant human ROR1 protein (Sino Biological Inc., Beijing, China).

    Techniques: Clone Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Marker, Staining, SDS Page, Western Blot, Expressing, Sonication, Transfection, Purification, Affinity Column