elisa kit  (Sino Biological)


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    SARS CoV 2 2019 nCoV Spike RBD Gene ORF cDNA clone expression plasmid C GFPSpark tag Codon Optimized COVID 19 Spike S RBD Research
    Description:
    Full length Clone DNA of SARS CoV 2 2019 nCoV Spike RBD
    Catalog Number:
    VG40592-ACG
    Price:
    215.0
    Category:
    cDNA Clone
    Applications:
    Stable or Transient mammalian expression
    Size:
    1Unit
    Product Aliases:
    coronavirus spike cDNA ORF Clone 2019-nCoV, cov spike cDNA ORF Clone 2019-nCoV, ncov RBD cDNA ORF Clone 2019-nCoV, ncov s1 cDNA ORF Clone 2019-nCoV, ncov s2 cDNA ORF Clone 2019-nCoV, ncov spike cDNA ORF Clone 2019-nCoV, NCP-CoV RBD cDNA ORF Clone 2019-nCoV, NCP-CoV s1 cDNA ORF Clone 2019-nCoV, NCP-CoV s2 cDNA ORF Clone 2019-nCoV, NCP-CoV Spike cDNA ORF Clone 2019-nCoV, novel coronavirus RBD cDNA ORF Clone 2019-nCoV, novel coronavirus s1 cDNA ORF Clone 2019-nCoV, novel coronavirus s2 cDNA ORF Clone 2019-nCoV, novel coronavirus spike cDNA ORF Clone 2019-nCoV, RBD cDNA ORF Clone 2019-nCoV, S1 cDNA ORF Clone 2019-nCoV, S2 cDNA ORF Clone 2019-nCoV, Spike RBD cDNA ORF Clone 2019-nCoV
    Molecule Name:
    Spike
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    Structured Review

    Sino Biological elisa kit
    SARS CoV 2 2019 nCoV Spike RBD Gene ORF cDNA clone expression plasmid C GFPSpark tag Codon Optimized COVID 19 Spike S RBD Research
    Full length Clone DNA of SARS CoV 2 2019 nCoV Spike RBD
    https://www.bioz.com/result/elisa kit/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    elisa kit - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Expanded antigen-experienced CD160+CD8+effector T cells exhibit impaired effector functions in chronic lymphocytic leukemia"

    Article Title: Expanded antigen-experienced CD160+CD8+effector T cells exhibit impaired effector functions in chronic lymphocytic leukemia

    Journal: Journal for Immunotherapy of Cancer

    doi: 10.1136/jitc-2020-002189

    The plasma cytokines/chemokines in the plasma of patients with chronic lymphocytic leukemia (CLL) versus healthy controls (HCs). (A) The volcano plot illustrating the magnitude and significance of differences in cytokine/chemokines plasma concentrations (measured by the Mesoplex assay) in patients with CLL versus HCs. (B) Scatter plot of the correlation between percentages of CD160 + CD8 + T cells in peripheral blood mononuclear cells (PBMCs) with the interleukin-16 (IL-16), and (C) MIP-1α concentrations in the plasma of patients with CLL. (D) Cumulative data showing IL-16 concentrations in the plasma of patients with CLL in low (0), intermediate (I/II), and high (III/IV) Rai stages, 16, 24 and 7 patients/group, respectively. (E) Cumulative data of percentages of CD160 + expressing cells among CD8 + T cells in PBMCs of patients with CLL in low (0), intermediate (I/II), and high (III/IV) Rai stages, 9, 20 and 6 patients/group, respectively. (F) Fold regulation of CD160 gene in CD8 + T cells stimulated with the anti-CD3/CD28 antibodies in the absence or presence of rh-IL-16 (500 ng/mL) for 72 hours relative to stimulated as quantified by qPCR from seven human subjects/group. (G) Cumulative data of IL-16 production in cell culture supernatants of isolated B-CLL versus non-B-CLLs after 12 hours’ culture as detected by ELISA from four patients. (H) Representative flow cytometry plots, and (I) cumulative data of intracytoplasmic IL-16 expression in CD8 + , CD4 + , and B cells of patients with CLL versus HCs. MIP-1α, macrophage inflammatory protein-1 alpha.
    Figure Legend Snippet: The plasma cytokines/chemokines in the plasma of patients with chronic lymphocytic leukemia (CLL) versus healthy controls (HCs). (A) The volcano plot illustrating the magnitude and significance of differences in cytokine/chemokines plasma concentrations (measured by the Mesoplex assay) in patients with CLL versus HCs. (B) Scatter plot of the correlation between percentages of CD160 + CD8 + T cells in peripheral blood mononuclear cells (PBMCs) with the interleukin-16 (IL-16), and (C) MIP-1α concentrations in the plasma of patients with CLL. (D) Cumulative data showing IL-16 concentrations in the plasma of patients with CLL in low (0), intermediate (I/II), and high (III/IV) Rai stages, 16, 24 and 7 patients/group, respectively. (E) Cumulative data of percentages of CD160 + expressing cells among CD8 + T cells in PBMCs of patients with CLL in low (0), intermediate (I/II), and high (III/IV) Rai stages, 9, 20 and 6 patients/group, respectively. (F) Fold regulation of CD160 gene in CD8 + T cells stimulated with the anti-CD3/CD28 antibodies in the absence or presence of rh-IL-16 (500 ng/mL) for 72 hours relative to stimulated as quantified by qPCR from seven human subjects/group. (G) Cumulative data of IL-16 production in cell culture supernatants of isolated B-CLL versus non-B-CLLs after 12 hours’ culture as detected by ELISA from four patients. (H) Representative flow cytometry plots, and (I) cumulative data of intracytoplasmic IL-16 expression in CD8 + , CD4 + , and B cells of patients with CLL versus HCs. MIP-1α, macrophage inflammatory protein-1 alpha.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    Plasma-derived extracellular vesicles (EVs) contain CD160. (A) Representative flow cytometry plots of CD160 expression in CD8 + and CD4 + T cells untreated versus treated with the plasma from patients with chronic lymphocytic leukemia (CCL) (using 5%, 10% and 20% plasma) after 72 hours of in vitro culture. (B) Cumulative data of percentages of CD160 expressing cells among CD8 + , and (C) CD4 + T cells either untreated or treated with indicated plasma concentrations after 72 hours. (D) Quantification of EV numbers isolated from the plasma of CLLs versus healthy controls (HCs) by Exocet ELISA kit. (E) ImageStream plots of plasma-derived EVs showing the expression of CD9, CD63 and CD160, bright field (BF). (F) ImageStream plots of plasma-derived EVs showing expression of CD63, CD81 and CD160. (G) Representative western blot (WB) images of plasma-derived EVs from HCs and patients with CLL depicting CD160 presence. (H) Cumulative data showing normalized arbitrary units of CD160/actin in plasma-derived EVs in HCs versus patients with CLL. (I) Representative WB images of plasma-derived EVs depicting CD9 expression, and (J) cumulative data showing normalized arbitrary units of CD9/actin in plasma-derived EVs in HCs versus patients with CLL. (K) Representative WB images of plasma-derived EVs depicting CD63 expression, and (L) cumulative data showing normalized arbitrary units of CD63/GAPDH in plasma-derived EVs in HCs versus patients with CLL. (M) Representative WB images of plasma-derived EVs depicting CD81 expression, and (N) cumulative data showing normalized arbitrary units of CD160/actin in plasma-derived EVs in HCs versus patients with CLL. Actin was used as a loading control to normalize protein amounts of CD81, CD9, and CD160, and GAPDH was used as a loading control to normalize protein amount of CD63. Each dot/band represents data from a subject.
    Figure Legend Snippet: Plasma-derived extracellular vesicles (EVs) contain CD160. (A) Representative flow cytometry plots of CD160 expression in CD8 + and CD4 + T cells untreated versus treated with the plasma from patients with chronic lymphocytic leukemia (CCL) (using 5%, 10% and 20% plasma) after 72 hours of in vitro culture. (B) Cumulative data of percentages of CD160 expressing cells among CD8 + , and (C) CD4 + T cells either untreated or treated with indicated plasma concentrations after 72 hours. (D) Quantification of EV numbers isolated from the plasma of CLLs versus healthy controls (HCs) by Exocet ELISA kit. (E) ImageStream plots of plasma-derived EVs showing the expression of CD9, CD63 and CD160, bright field (BF). (F) ImageStream plots of plasma-derived EVs showing expression of CD63, CD81 and CD160. (G) Representative western blot (WB) images of plasma-derived EVs from HCs and patients with CLL depicting CD160 presence. (H) Cumulative data showing normalized arbitrary units of CD160/actin in plasma-derived EVs in HCs versus patients with CLL. (I) Representative WB images of plasma-derived EVs depicting CD9 expression, and (J) cumulative data showing normalized arbitrary units of CD9/actin in plasma-derived EVs in HCs versus patients with CLL. (K) Representative WB images of plasma-derived EVs depicting CD63 expression, and (L) cumulative data showing normalized arbitrary units of CD63/GAPDH in plasma-derived EVs in HCs versus patients with CLL. (M) Representative WB images of plasma-derived EVs depicting CD81 expression, and (N) cumulative data showing normalized arbitrary units of CD160/actin in plasma-derived EVs in HCs versus patients with CLL. Actin was used as a loading control to normalize protein amounts of CD81, CD9, and CD160, and GAPDH was used as a loading control to normalize protein amount of CD63. Each dot/band represents data from a subject.

    Techniques Used: Derivative Assay, Flow Cytometry, Expressing, In Vitro, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot

    2) Product Images from "BAMBI and CHGA in Prion Diseases: Neuropathological Assessment and Potential Role as Disease Biomarkers"

    Article Title: BAMBI and CHGA in Prion Diseases: Neuropathological Assessment and Potential Role as Disease Biomarkers

    Journal: Biomolecules

    doi: 10.3390/biom10050706

    ELISA quantification of BAMBI and CHGA proteins in CSF of scrapie-infected sheep and BAMBI in CSF of CJD patients. ( A ) Intensity of BAMBI and CHGA signals in CSF of control (NC, round dots) and scrapie sheep at preclinical (PS, triangular dots) and clinical stage (CS, square dots). Protein BAMBI displayed an upregulation in CSF of scrapie-affected sheep at clinical stage (2465 ± 174 pg/mL) compared to scrapie sheep at preclinical stage (2103 ± 132 pg/mL, p
    Figure Legend Snippet: ELISA quantification of BAMBI and CHGA proteins in CSF of scrapie-infected sheep and BAMBI in CSF of CJD patients. ( A ) Intensity of BAMBI and CHGA signals in CSF of control (NC, round dots) and scrapie sheep at preclinical (PS, triangular dots) and clinical stage (CS, square dots). Protein BAMBI displayed an upregulation in CSF of scrapie-affected sheep at clinical stage (2465 ± 174 pg/mL) compared to scrapie sheep at preclinical stage (2103 ± 132 pg/mL, p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Infection

    3) Product Images from "Integrative gene ontology and network analysis of coronary artery disease associated genes suggests potential role of ErbB pathway gene EGFR"

    Article Title: Integrative gene ontology and network analysis of coronary artery disease associated genes suggests potential role of ErbB pathway gene EGFR

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.8393

    EGFR levels confirmed by western blot analysis and ELISA in CAD patients and control subjects. (A) Western blotting images of plasma EGFR and loading control GAPDH were assayed in CAD patients and controls. (B) Quantification of relative intensity levels of EGFR in CAD patients and controls adjusted for loading control. ImageJ software was used. (C) ELISA-based comparison of mean EGFR levels between the CAD patients and control groups. A significantly higher level of EGFR was observed in the MI and SA groups in comparison with controls. (D) EGFR levels were only significantly different between male MI groups in comparison with male control group. The EGFR levels were significantly different between female MI and SA groups when compared with controls. *P≤0.05, **P≤0.001. EGFR, epidermal growth factor receptor; CAD, coronary artery disease; MI, myocardial infarction; SA, stable angina.
    Figure Legend Snippet: EGFR levels confirmed by western blot analysis and ELISA in CAD patients and control subjects. (A) Western blotting images of plasma EGFR and loading control GAPDH were assayed in CAD patients and controls. (B) Quantification of relative intensity levels of EGFR in CAD patients and controls adjusted for loading control. ImageJ software was used. (C) ELISA-based comparison of mean EGFR levels between the CAD patients and control groups. A significantly higher level of EGFR was observed in the MI and SA groups in comparison with controls. (D) EGFR levels were only significantly different between male MI groups in comparison with male control group. The EGFR levels were significantly different between female MI and SA groups when compared with controls. *P≤0.05, **P≤0.001. EGFR, epidermal growth factor receptor; CAD, coronary artery disease; MI, myocardial infarction; SA, stable angina.

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Software

    An overview of the biomarker prioritization method. The method of biomarker prioritization involves the following: Step 1, selection of CAD related genes from CADgene database; Step 2, gene filtration based on GO and DO; Step 3, the gene set obtained was used for network construction, analysis, identification of hub and pathway enrichment analysis; and, Step 4, biological validation using WB, ELISA and statistical assessment of the biomarker. GO, gene ontology; DO, disease ontology; WB, western blot analysis; CAD, coronary artery disease; EGFR, epidermal growth factor receptor.
    Figure Legend Snippet: An overview of the biomarker prioritization method. The method of biomarker prioritization involves the following: Step 1, selection of CAD related genes from CADgene database; Step 2, gene filtration based on GO and DO; Step 3, the gene set obtained was used for network construction, analysis, identification of hub and pathway enrichment analysis; and, Step 4, biological validation using WB, ELISA and statistical assessment of the biomarker. GO, gene ontology; DO, disease ontology; WB, western blot analysis; CAD, coronary artery disease; EGFR, epidermal growth factor receptor.

    Techniques Used: Biomarker Assay, Selection, Filtration, Western Blot, Enzyme-linked Immunosorbent Assay

    4) Product Images from "VHL mutation-mediated SALL4 overexpression promotes tumorigenesis and vascularization of clear cell renal cell carcinoma via Akt/GSK-3β signaling"

    Article Title: VHL mutation-mediated SALL4 overexpression promotes tumorigenesis and vascularization of clear cell renal cell carcinoma via Akt/GSK-3β signaling

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-020-01609-8

    SALL4 induces ccRCC angiogenesis in vitro. a Enriched gene ontology (GO) term and KEGG pathway analysis of genes significantly correlated with SALL4 in ccRCC. b , c Gene set enrichment analysis (GSEA) profiles for the genes significantly correlated with SALL4 in ccRCC. Data ( a - c ) from TCGA database were analyzed via LinkedOmics bioinformatics. d HUVECs were treated with serum-free medium (SFM) or conditioned medium (CMs) from ACHN-shNC/shSALL4 cells. After treatment for 72 h, CCK-8 assays were performed to evaluate cell proliferation. e Flow cytometry analyses of cell cycle distribution in HUVECs treated with SFM or CMs from ACHN-shNC/shSALL4 cells. f Wound-healing assay was performed to determine the migration of HUVECs with CMs treatment (scale bar, 200 μm). g Representative images and quantification analyses of the recruitment of HUVECs co-cultured with indicated CMs ( upper ) or ACHN-shNC/shSALL4 cells ( lower ) in lower chambers (scale bar, 100 μm). h, i Representative images ( h ) and quantification analyses ( i ) of tube formation in HUVECs treated with SFM or indicated CMs (scale bar, 100 μm). j The expression level of VEGFA in CMs from ACHN-shNC/shSALL4 cells was detected by ELISA assay. * P
    Figure Legend Snippet: SALL4 induces ccRCC angiogenesis in vitro. a Enriched gene ontology (GO) term and KEGG pathway analysis of genes significantly correlated with SALL4 in ccRCC. b , c Gene set enrichment analysis (GSEA) profiles for the genes significantly correlated with SALL4 in ccRCC. Data ( a - c ) from TCGA database were analyzed via LinkedOmics bioinformatics. d HUVECs were treated with serum-free medium (SFM) or conditioned medium (CMs) from ACHN-shNC/shSALL4 cells. After treatment for 72 h, CCK-8 assays were performed to evaluate cell proliferation. e Flow cytometry analyses of cell cycle distribution in HUVECs treated with SFM or CMs from ACHN-shNC/shSALL4 cells. f Wound-healing assay was performed to determine the migration of HUVECs with CMs treatment (scale bar, 200 μm). g Representative images and quantification analyses of the recruitment of HUVECs co-cultured with indicated CMs ( upper ) or ACHN-shNC/shSALL4 cells ( lower ) in lower chambers (scale bar, 100 μm). h, i Representative images ( h ) and quantification analyses ( i ) of tube formation in HUVECs treated with SFM or indicated CMs (scale bar, 100 μm). j The expression level of VEGFA in CMs from ACHN-shNC/shSALL4 cells was detected by ELISA assay. * P

    Techniques Used: In Vitro, CCK-8 Assay, Flow Cytometry, Wound Healing Assay, Migration, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Suppression of R5-type of HIV-1 in CD4+ NKT cells by Vδ1+ T cells activated by flavonoid glycosides, hesperidin and linarin"

    Article Title: Suppression of R5-type of HIV-1 in CD4+ NKT cells by Vδ1+ T cells activated by flavonoid glycosides, hesperidin and linarin

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-40587-6

    Effects of hesperidin and linarin on the replication of R5-type of HIV-1 in CD4 + NKT cells through Vδ1 + T cells. ( A ) CD4 + NKT cells induced from PBMCs by using α-GalCer (left). Viability of CD4 + NKT cells cultured with hesperidin, linarin or DMSO (right). ( B ) As described in Methods, CD4 + NKT cells were infected with R5-tropic NL(AD8) HIV-1. Then, HIV-1-infected CD4 + NKT cells were cultured in the presence of 3, 10, or 30 μg/mL hesperidin or linarin or 0.003, 0.01 or 0.03% DMSO with (right)/without (left) the resting Vδ1 + T cells for 3 days. HIV-1 p24 concentration of the culture supernatant was measured by using specific ELISA kit. ( C ) The percentage of HIV-1 viral replication was also shown. The data are expressed as the mean + SEM of three independent experiments. * P
    Figure Legend Snippet: Effects of hesperidin and linarin on the replication of R5-type of HIV-1 in CD4 + NKT cells through Vδ1 + T cells. ( A ) CD4 + NKT cells induced from PBMCs by using α-GalCer (left). Viability of CD4 + NKT cells cultured with hesperidin, linarin or DMSO (right). ( B ) As described in Methods, CD4 + NKT cells were infected with R5-tropic NL(AD8) HIV-1. Then, HIV-1-infected CD4 + NKT cells were cultured in the presence of 3, 10, or 30 μg/mL hesperidin or linarin or 0.003, 0.01 or 0.03% DMSO with (right)/without (left) the resting Vδ1 + T cells for 3 days. HIV-1 p24 concentration of the culture supernatant was measured by using specific ELISA kit. ( C ) The percentage of HIV-1 viral replication was also shown. The data are expressed as the mean + SEM of three independent experiments. * P

    Techniques Used: Cell Culture, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay

    6) Product Images from "Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway"

    Article Title: Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway

    Journal: Diabetes

    doi: 10.2337/db12-1710

    Kallistatin specifically binds to the extracellular domain of LRP6 with high affinity. A : The human serum was incubated with the conditioned medium expressing LRP6N-Myc followed by immunoprecipitation using the anti–Myc-tag antibody–conjugated resin. Precipitates and input samples were immunoblotted (IB) with anti-kallistatin (KS), -albumin, and –Myc-tag antibodies. B : Immunoprecipitation was performed with the Ni-NTA resin in LRP6N-Myc conditioned medium with or without recombinant KS-HIS. Precipitates and input samples were immunoblotted with anti–Myc-tag and anti–HIS-tag antibodies to detect LRP6N-Myc and KS-HIS protein. C and D : Negative control for coimmunoprecipitation. Immunoprecipitation was performed with the Ni-NTA resin (Novagen, Madison, WI) in Fz8CRD-IgG (CRD domain of Fz-8 with human IgG tag) and LDLRN-Myc–conditioned medium incubated with recombinant KS-HIS. Precipitates and input samples were immunoblotted with antibodies as indicated to identify Fz8CRD-IgG, KS-HIS, and LDLRN-Myc proteins in the precipitates. E : ELISA plates were coated with conditioned medium of LRP6N and LDLRN (negative control) overnight. Different concentrations of purified kallistatin were incubated in the wells. The signals were developed using a Biotin-labeled secondary antibody for kallistatin and measured by absorbance at 450 nm using a microplate reader. The binding value at each concentration was plotted after subtraction of that of nonspecific binding to LDLRN (mean ± SD, n = 5).
    Figure Legend Snippet: Kallistatin specifically binds to the extracellular domain of LRP6 with high affinity. A : The human serum was incubated with the conditioned medium expressing LRP6N-Myc followed by immunoprecipitation using the anti–Myc-tag antibody–conjugated resin. Precipitates and input samples were immunoblotted (IB) with anti-kallistatin (KS), -albumin, and –Myc-tag antibodies. B : Immunoprecipitation was performed with the Ni-NTA resin in LRP6N-Myc conditioned medium with or without recombinant KS-HIS. Precipitates and input samples were immunoblotted with anti–Myc-tag and anti–HIS-tag antibodies to detect LRP6N-Myc and KS-HIS protein. C and D : Negative control for coimmunoprecipitation. Immunoprecipitation was performed with the Ni-NTA resin (Novagen, Madison, WI) in Fz8CRD-IgG (CRD domain of Fz-8 with human IgG tag) and LDLRN-Myc–conditioned medium incubated with recombinant KS-HIS. Precipitates and input samples were immunoblotted with antibodies as indicated to identify Fz8CRD-IgG, KS-HIS, and LDLRN-Myc proteins in the precipitates. E : ELISA plates were coated with conditioned medium of LRP6N and LDLRN (negative control) overnight. Different concentrations of purified kallistatin were incubated in the wells. The signals were developed using a Biotin-labeled secondary antibody for kallistatin and measured by absorbance at 450 nm using a microplate reader. The binding value at each concentration was plotted after subtraction of that of nonspecific binding to LDLRN (mean ± SD, n = 5).

    Techniques Used: Incubation, Expressing, Immunoprecipitation, Recombinant, Negative Control, Enzyme-linked Immunosorbent Assay, Purification, Labeling, Binding Assay, Concentration Assay

    Decreased retinal neuroinflammation and retinal vascular leakage in diabetic kallistatin-TG mice. Akita mice and Akita×kallistatin-TG mice at 5 months of age were used for leukostasis assay. A : Adherent leukocytes (arrow) were stained with FITC-conjugated concanavalin-A in the retinal vasculature. The retinal vasculature and leukocytes were visualized in retinal flat mounts under a fluorescence microscope. Scale bar: 50 μm. B : Adherent leukocytes in the retinal vasculature were counted in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice. n = 7–10. C : Retinal levels of soluble ICAM-1 were measured by ELISA in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. D : Retinal vascular permeability of WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice was measured using Evans blue as a tracer, normalized by retinal protein concentrations, and expressed as a percentage of the permeability in WT control. E : Retinal levels of VEGF-A were measured by ELISA in WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. All values are mean ± SD. * P
    Figure Legend Snippet: Decreased retinal neuroinflammation and retinal vascular leakage in diabetic kallistatin-TG mice. Akita mice and Akita×kallistatin-TG mice at 5 months of age were used for leukostasis assay. A : Adherent leukocytes (arrow) were stained with FITC-conjugated concanavalin-A in the retinal vasculature. The retinal vasculature and leukocytes were visualized in retinal flat mounts under a fluorescence microscope. Scale bar: 50 μm. B : Adherent leukocytes in the retinal vasculature were counted in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice. n = 7–10. C : Retinal levels of soluble ICAM-1 were measured by ELISA in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. D : Retinal vascular permeability of WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice was measured using Evans blue as a tracer, normalized by retinal protein concentrations, and expressed as a percentage of the permeability in WT control. E : Retinal levels of VEGF-A were measured by ELISA in WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. All values are mean ± SD. * P

    Techniques Used: Mouse Assay, Staining, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Permeability

    Alleviated retinal neovascularization and retinal neuroinflammation in the kallistatin-TG mice with OIR. Kallistatin-TG and WT mice were exposed to 75% oxygen from P7 to P12. Angiography was performed using FITC-dextran at P18 and with the retina flat mounted. A : Representative retinal angiographs from the eyes of WT and kallistatin-TG mice with OIR. The white arrows indicate neovascular tufts in the retina. The white dotted line outlines a vascular area in the central retinal. Scale bar: 100 μm. B : Quantification of nonperfused area. Retinal nonperfused area was semiquantified using ImageJ software and expressed as % of the whole area of the retina. n = 6–9. At P18, the eyes of kallistatin-TG and WT mice with OIR were fixed, sectioned, and stained with H-E. C : Representative retinal sections from WT and kallistatin-TG mice with OIR. Arrows indicate preretinal vascular cells. Scale bar: 50 μm. D : Preretinal vascular cells were quantified and compared between WT and kallistatin-TG mice with OIR. n = 8. E : Retinal levels of VEGF and ICAM-1 were measured with ELISA and expressed as percentages of the respective WT control. n = 8. All values are mean ± SD. * P
    Figure Legend Snippet: Alleviated retinal neovascularization and retinal neuroinflammation in the kallistatin-TG mice with OIR. Kallistatin-TG and WT mice were exposed to 75% oxygen from P7 to P12. Angiography was performed using FITC-dextran at P18 and with the retina flat mounted. A : Representative retinal angiographs from the eyes of WT and kallistatin-TG mice with OIR. The white arrows indicate neovascular tufts in the retina. The white dotted line outlines a vascular area in the central retinal. Scale bar: 100 μm. B : Quantification of nonperfused area. Retinal nonperfused area was semiquantified using ImageJ software and expressed as % of the whole area of the retina. n = 6–9. At P18, the eyes of kallistatin-TG and WT mice with OIR were fixed, sectioned, and stained with H-E. C : Representative retinal sections from WT and kallistatin-TG mice with OIR. Arrows indicate preretinal vascular cells. Scale bar: 50 μm. D : Preretinal vascular cells were quantified and compared between WT and kallistatin-TG mice with OIR. n = 8. E : Retinal levels of VEGF and ICAM-1 were measured with ELISA and expressed as percentages of the respective WT control. n = 8. All values are mean ± SD. * P

    Techniques Used: Mouse Assay, Software, Staining, Enzyme-linked Immunosorbent Assay

    7) Product Images from "Identification of Fucosylated SERPINA1 as a Novel Plasma Marker for Pancreatic Cancer Using Lectin Affinity Capture Coupled with iTRAQ-Based Quantitative Glycoproteomics"

    Article Title: Identification of Fucosylated SERPINA1 as a Novel Plasma Marker for Pancreatic Cancer Using Lectin Affinity Capture Coupled with iTRAQ-Based Quantitative Glycoproteomics

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22116079

    Plasma levels of fuco-SERPINA1, SERPINA1 and CA19-9 measured in enrolled subjects. ( a , b ) Levels of fuco-SERPINA1 measured in plasma specimens from GS and PC patients using AAL-based reverse lectin ELISA. ( c , d ) Levels of SERPINA1 protein measured in plasma specimens from GS and PC patients using commercial ELISA kits. ( e , f ) Levels of CA19-9 measured in plasma specimens from GS and PC patients using ECLIA. The horizontal lines indicate mean ± S.D. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
    Figure Legend Snippet: Plasma levels of fuco-SERPINA1, SERPINA1 and CA19-9 measured in enrolled subjects. ( a , b ) Levels of fuco-SERPINA1 measured in plasma specimens from GS and PC patients using AAL-based reverse lectin ELISA. ( c , d ) Levels of SERPINA1 protein measured in plasma specimens from GS and PC patients using commercial ELISA kits. ( e , f ) Levels of CA19-9 measured in plasma specimens from GS and PC patients using ECLIA. The horizontal lines indicate mean ± S.D. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Schematic representation of the experimental design of this study. iTRAQ labeling coupled with 2D-SCX/RP-LC-MS/MS was applied for comprehensive analysis of the proteome profile of plasma samples from patients with PC, including non-metastatic (M0) and metastatic (M1) pancreatic cancers and subjects with gallstone lesions (GS). AAL was employed to enrich glycopeptides with specific glycan and H 2 18 O to label glycosylated sites via PNGase F-mediated reaction for glycoproteome profiling analysis. We selected 18 O-labeled fucose-containing glycopeptides upregulated in PC plasma samples as candidate targets. Fuco-SERPINA1, one of the candidate proteins, was selected for validation in plasma samples of PC and GS using reverse AAL-based ELISA.
    Figure Legend Snippet: Schematic representation of the experimental design of this study. iTRAQ labeling coupled with 2D-SCX/RP-LC-MS/MS was applied for comprehensive analysis of the proteome profile of plasma samples from patients with PC, including non-metastatic (M0) and metastatic (M1) pancreatic cancers and subjects with gallstone lesions (GS). AAL was employed to enrich glycopeptides with specific glycan and H 2 18 O to label glycosylated sites via PNGase F-mediated reaction for glycoproteome profiling analysis. We selected 18 O-labeled fucose-containing glycopeptides upregulated in PC plasma samples as candidate targets. Fuco-SERPINA1, one of the candidate proteins, was selected for validation in plasma samples of PC and GS using reverse AAL-based ELISA.

    Techniques Used: Labeling, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

    Evaluation of the specificity of AAL binding to fuco-SERPINA1 in reverse lectin ELISA. ( a ) Different amounts of recombinant SERPINA1 protein were treated with or without PNGase F (PNG) (protein:PNGase F = 1 µg:1U, 37 °C for 20 h) and subjected to AAL-based reverse lectin ELISA. ( b ) AAL was pre-incubated with different doses of L-fucose or lactose at room temperature for 0.5 h and coated onto ELISA plates. Plates were subjected to reverse lectin ELISA for detecting fixed amounts of fuco-SERPINA1. OD values were measured at 450 nm and each data point was examined in duplicate. Data expressed as mean ± S.D. are presented as a black solid line.
    Figure Legend Snippet: Evaluation of the specificity of AAL binding to fuco-SERPINA1 in reverse lectin ELISA. ( a ) Different amounts of recombinant SERPINA1 protein were treated with or without PNGase F (PNG) (protein:PNGase F = 1 µg:1U, 37 °C for 20 h) and subjected to AAL-based reverse lectin ELISA. ( b ) AAL was pre-incubated with different doses of L-fucose or lactose at room temperature for 0.5 h and coated onto ELISA plates. Plates were subjected to reverse lectin ELISA for detecting fixed amounts of fuco-SERPINA1. OD values were measured at 450 nm and each data point was examined in duplicate. Data expressed as mean ± S.D. are presented as a black solid line.

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

    8) Product Images from "Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway"

    Article Title: Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway

    Journal: Diabetes

    doi: 10.2337/db12-1710

    Kallistatin specifically binds to the extracellular domain of LRP6 with high affinity. A : The human serum was incubated with the conditioned medium expressing LRP6N-Myc followed by immunoprecipitation using the anti–Myc-tag antibody–conjugated resin. Precipitates and input samples were immunoblotted (IB) with anti-kallistatin (KS), -albumin, and –Myc-tag antibodies. B : Immunoprecipitation was performed with the Ni-NTA resin in LRP6N-Myc conditioned medium with or without recombinant KS-HIS. Precipitates and input samples were immunoblotted with anti–Myc-tag and anti–HIS-tag antibodies to detect LRP6N-Myc and KS-HIS protein. C and D : Negative control for coimmunoprecipitation. Immunoprecipitation was performed with the Ni-NTA resin (Novagen, Madison, WI) in Fz8CRD-IgG (CRD domain of Fz-8 with human IgG tag) and LDLRN-Myc–conditioned medium incubated with recombinant KS-HIS. Precipitates and input samples were immunoblotted with antibodies as indicated to identify Fz8CRD-IgG, KS-HIS, and LDLRN-Myc proteins in the precipitates. E : ELISA plates were coated with conditioned medium of LRP6N and LDLRN (negative control) overnight. Different concentrations of purified kallistatin were incubated in the wells. The signals were developed using a Biotin-labeled secondary antibody for kallistatin and measured by absorbance at 450 nm using a microplate reader. The binding value at each concentration was plotted after subtraction of that of nonspecific binding to LDLRN (mean ± SD, n = 5).
    Figure Legend Snippet: Kallistatin specifically binds to the extracellular domain of LRP6 with high affinity. A : The human serum was incubated with the conditioned medium expressing LRP6N-Myc followed by immunoprecipitation using the anti–Myc-tag antibody–conjugated resin. Precipitates and input samples were immunoblotted (IB) with anti-kallistatin (KS), -albumin, and –Myc-tag antibodies. B : Immunoprecipitation was performed with the Ni-NTA resin in LRP6N-Myc conditioned medium with or without recombinant KS-HIS. Precipitates and input samples were immunoblotted with anti–Myc-tag and anti–HIS-tag antibodies to detect LRP6N-Myc and KS-HIS protein. C and D : Negative control for coimmunoprecipitation. Immunoprecipitation was performed with the Ni-NTA resin (Novagen, Madison, WI) in Fz8CRD-IgG (CRD domain of Fz-8 with human IgG tag) and LDLRN-Myc–conditioned medium incubated with recombinant KS-HIS. Precipitates and input samples were immunoblotted with antibodies as indicated to identify Fz8CRD-IgG, KS-HIS, and LDLRN-Myc proteins in the precipitates. E : ELISA plates were coated with conditioned medium of LRP6N and LDLRN (negative control) overnight. Different concentrations of purified kallistatin were incubated in the wells. The signals were developed using a Biotin-labeled secondary antibody for kallistatin and measured by absorbance at 450 nm using a microplate reader. The binding value at each concentration was plotted after subtraction of that of nonspecific binding to LDLRN (mean ± SD, n = 5).

    Techniques Used: Incubation, Expressing, Immunoprecipitation, Recombinant, Negative Control, Enzyme-linked Immunosorbent Assay, Purification, Labeling, Binding Assay, Concentration Assay

    Decreased retinal neuroinflammation and retinal vascular leakage in diabetic kallistatin-TG mice. Akita mice and Akita×kallistatin-TG mice at 5 months of age were used for leukostasis assay. A : Adherent leukocytes (arrow) were stained with FITC-conjugated concanavalin-A in the retinal vasculature. The retinal vasculature and leukocytes were visualized in retinal flat mounts under a fluorescence microscope. Scale bar: 50 μm. B : Adherent leukocytes in the retinal vasculature were counted in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice. n = 7–10. C : Retinal levels of soluble ICAM-1 were measured by ELISA in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. D : Retinal vascular permeability of WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice was measured using Evans blue as a tracer, normalized by retinal protein concentrations, and expressed as a percentage of the permeability in WT control. E : Retinal levels of VEGF-A were measured by ELISA in WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. All values are mean ± SD. * P
    Figure Legend Snippet: Decreased retinal neuroinflammation and retinal vascular leakage in diabetic kallistatin-TG mice. Akita mice and Akita×kallistatin-TG mice at 5 months of age were used for leukostasis assay. A : Adherent leukocytes (arrow) were stained with FITC-conjugated concanavalin-A in the retinal vasculature. The retinal vasculature and leukocytes were visualized in retinal flat mounts under a fluorescence microscope. Scale bar: 50 μm. B : Adherent leukocytes in the retinal vasculature were counted in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice. n = 7–10. C : Retinal levels of soluble ICAM-1 were measured by ELISA in age-matched WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. D : Retinal vascular permeability of WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice was measured using Evans blue as a tracer, normalized by retinal protein concentrations, and expressed as a percentage of the permeability in WT control. E : Retinal levels of VEGF-A were measured by ELISA in WT, kallistatin-TG, Akita, and Akita×kallistatin-TG mice and expressed as percentages of the respective WT control. All values are mean ± SD. * P

    Techniques Used: Mouse Assay, Staining, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Permeability

    Alleviated retinal neovascularization and retinal neuroinflammation in the kallistatin-TG mice with OIR. Kallistatin-TG and WT mice were exposed to 75% oxygen from P7 to P12. Angiography was performed using FITC-dextran at P18 and with the retina flat mounted. A : Representative retinal angiographs from the eyes of WT and kallistatin-TG mice with OIR. The white arrows indicate neovascular tufts in the retina. The white dotted line outlines a vascular area in the central retinal. Scale bar: 100 μm. B : Quantification of nonperfused area. Retinal nonperfused area was semiquantified using ImageJ software and expressed as % of the whole area of the retina. n = 6–9. At P18, the eyes of kallistatin-TG and WT mice with OIR were fixed, sectioned, and stained with H-E. C : Representative retinal sections from WT and kallistatin-TG mice with OIR. Arrows indicate preretinal vascular cells. Scale bar: 50 μm. D : Preretinal vascular cells were quantified and compared between WT and kallistatin-TG mice with OIR. n = 8. E : Retinal levels of VEGF and ICAM-1 were measured with ELISA and expressed as percentages of the respective WT control. n = 8. All values are mean ± SD. * P
    Figure Legend Snippet: Alleviated retinal neovascularization and retinal neuroinflammation in the kallistatin-TG mice with OIR. Kallistatin-TG and WT mice were exposed to 75% oxygen from P7 to P12. Angiography was performed using FITC-dextran at P18 and with the retina flat mounted. A : Representative retinal angiographs from the eyes of WT and kallistatin-TG mice with OIR. The white arrows indicate neovascular tufts in the retina. The white dotted line outlines a vascular area in the central retinal. Scale bar: 100 μm. B : Quantification of nonperfused area. Retinal nonperfused area was semiquantified using ImageJ software and expressed as % of the whole area of the retina. n = 6–9. At P18, the eyes of kallistatin-TG and WT mice with OIR were fixed, sectioned, and stained with H-E. C : Representative retinal sections from WT and kallistatin-TG mice with OIR. Arrows indicate preretinal vascular cells. Scale bar: 50 μm. D : Preretinal vascular cells were quantified and compared between WT and kallistatin-TG mice with OIR. n = 8. E : Retinal levels of VEGF and ICAM-1 were measured with ELISA and expressed as percentages of the respective WT control. n = 8. All values are mean ± SD. * P

    Techniques Used: Mouse Assay, Software, Staining, Enzyme-linked Immunosorbent Assay

    9) Product Images from "Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis"

    Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-021-03545-7

    Expression of CXCL11 in tissue samples and cancer-associated fibroblasts (CAFs). A CAFs and normal fibroblasts were isolated from cancer tissues and noncancerous normal tissues, respectively, and identified for α-SMA protein content and distribution by Immunofluorescent (IF) staining. The relative fluorescence intensity was shown in the right panel. N = 5. B The mRNA expression of CXCL13, CXCL12, CCL2, CXCL11, CXCL16, CCL8, CXCL26, CXCL5, CXCL14, CCL5, and CCL7 was examined in CAFs and NFs using real-time PCR. C The protein levels of CXCL11 were examined in CAFs and NFs using Immunoblotting. D The contents of CXCL11 in the culture medium were determined by ELISA. E The protein levels of CXCL11 were examined in 12 cases of para-carcinoma tissues, 12 cases of nonmetastatic HCC tissues, and 12 cases of metastatic HCC tissues using Immunoblotting. F The mRNA expression of CXCL11 was examined in 12 cases of para-carcinoma tissues, 12 cases of nonmetastatic HCC tissues, and 12 cases of metastatic HCC tissues using real-time PCR. G The histopathological features of para-carcinoma tissues, nonmetastatic HCC tissues, and metastatic HCC tissues were examined by H E staining. The protein contents of CXCL11 in para-carcinoma tissues, nonmetastatic HCC tissues, and metastatic HCC tissues were examined by Immunohistochemical (IHC) staining. * P
    Figure Legend Snippet: Expression of CXCL11 in tissue samples and cancer-associated fibroblasts (CAFs). A CAFs and normal fibroblasts were isolated from cancer tissues and noncancerous normal tissues, respectively, and identified for α-SMA protein content and distribution by Immunofluorescent (IF) staining. The relative fluorescence intensity was shown in the right panel. N = 5. B The mRNA expression of CXCL13, CXCL12, CCL2, CXCL11, CXCL16, CCL8, CXCL26, CXCL5, CXCL14, CCL5, and CCL7 was examined in CAFs and NFs using real-time PCR. C The protein levels of CXCL11 were examined in CAFs and NFs using Immunoblotting. D The contents of CXCL11 in the culture medium were determined by ELISA. E The protein levels of CXCL11 were examined in 12 cases of para-carcinoma tissues, 12 cases of nonmetastatic HCC tissues, and 12 cases of metastatic HCC tissues using Immunoblotting. F The mRNA expression of CXCL11 was examined in 12 cases of para-carcinoma tissues, 12 cases of nonmetastatic HCC tissues, and 12 cases of metastatic HCC tissues using real-time PCR. G The histopathological features of para-carcinoma tissues, nonmetastatic HCC tissues, and metastatic HCC tissues were examined by H E staining. The protein contents of CXCL11 in para-carcinoma tissues, nonmetastatic HCC tissues, and metastatic HCC tissues were examined by Immunohistochemical (IHC) staining. * P

    Techniques Used: Expressing, Isolation, Staining, Fluorescence, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

    The function of circUBAP2 in the effects of CAFs-derived CXCL11 on HCC cells and the selection of mRNA related to the functions of circUBAP2. A Silencing of circUBAP2 was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting circUBAP2 (si-circUBAP2-1 or si-circUBAP2-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-circUBAP2-1 was chosen for further experiments for its better transfection efficiency. Then, MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for B Cell migration by Transwell assay; C Cell migration by wound healing assay; D The protein levels of Vimentin and Twist was examined by Immunoblotting; E The concentrations of IL-1β and IL-17 in the culture medium by ELISA. F , G The Volcano plot and hierarchical clustering heatmap showing differentially expressed mRNAs in CXCL11-treated MHCC-97H based on RNA sequencing. Upregulated genes were applied for H Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling enrichment analysis and I Gene Ontology (GO) of biological process enrichment analysis. J The expression of IFIT1/IFIT3 in CXCL11-treated and untreated HCC cells based on RNA sequencing data. * P
    Figure Legend Snippet: The function of circUBAP2 in the effects of CAFs-derived CXCL11 on HCC cells and the selection of mRNA related to the functions of circUBAP2. A Silencing of circUBAP2 was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting circUBAP2 (si-circUBAP2-1 or si-circUBAP2-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-circUBAP2-1 was chosen for further experiments for its better transfection efficiency. Then, MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for B Cell migration by Transwell assay; C Cell migration by wound healing assay; D The protein levels of Vimentin and Twist was examined by Immunoblotting; E The concentrations of IL-1β and IL-17 in the culture medium by ELISA. F , G The Volcano plot and hierarchical clustering heatmap showing differentially expressed mRNAs in CXCL11-treated MHCC-97H based on RNA sequencing. Upregulated genes were applied for H Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling enrichment analysis and I Gene Ontology (GO) of biological process enrichment analysis. J The expression of IFIT1/IFIT3 in CXCL11-treated and untreated HCC cells based on RNA sequencing data. * P

    Techniques Used: Derivative Assay, Selection, Small Interfering RNA, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Migration, Transwell Assay, Wound Healing Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Expressing

    10) Product Images from "Nonspreading Rift Valley Fever Virus Infection of Human Dendritic Cells Results in Downregulation of CD83 and Full Maturation of Bystander Cells"

    Article Title: Nonspreading Rift Valley Fever Virus Infection of Human Dendritic Cells Results in Downregulation of CD83 and Full Maturation of Bystander Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142670

    Effect of NSR infection on intracellular and extracellular CD83 levels. (A) The levels of soluble CD83 in supernatants from cells harvested 24 h after stimulation with LPS, infection with NSR, or from cells mock infected with NSRmock were determined by ELISA. Bars represent average CD83 concentrations ±SD. Results from one of two independently performed experiments with similar results are shown. (B) Detection of CD83 in cell lysates by Western blot at 24 hpi. The different treatments are shown above the top panel and the probed proteins are depicted at the right. The positions of molecular weight standard proteins are shown at the left. The top blot was stripped and re-probed with antibodies against GAPDH and GFP, which served as loading control and control to confirm NSR infection, respectively. Results from one of two independent experiments with cells from two donors are shown.
    Figure Legend Snippet: Effect of NSR infection on intracellular and extracellular CD83 levels. (A) The levels of soluble CD83 in supernatants from cells harvested 24 h after stimulation with LPS, infection with NSR, or from cells mock infected with NSRmock were determined by ELISA. Bars represent average CD83 concentrations ±SD. Results from one of two independently performed experiments with similar results are shown. (B) Detection of CD83 in cell lysates by Western blot at 24 hpi. The different treatments are shown above the top panel and the probed proteins are depicted at the right. The positions of molecular weight standard proteins are shown at the left. The top blot was stripped and re-probed with antibodies against GAPDH and GFP, which served as loading control and control to confirm NSR infection, respectively. Results from one of two independent experiments with cells from two donors are shown.

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Molecular Weight

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    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity
    Article Snippet: Our study will be helpful for understanding SARS-CoV-2 transmission and for the design of vaccines and therapeutic interventions against COVID-19. .. Plasmids The codon-optimized gene encoding SARS-CoV-2 S protein (GenBank: QHD43416) with C-terminal 19-amino acid deletion was synthesized by Sino Biological Inc (Beijing, China), and cloned into the Kpn I and Xba I restriction sites of pCMV3 vector (pCMV3-SARS-CoV-2-S-C19del, denoted as pS-D614). .. The D614G mutant S-expressing plasmid (denoted as pS-G614) was constructed by site-directed mutagenesis, with pS-D614 plasmid as a template.

    Article Title: Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies
    Article Snippet: .. DNA sequences encoding the reference strain wild-type (WT) and mutant Spike proteins of SARS-CoV-2 were codon-optimized and synthesized by Sino Biological Inc. (Beijing, China) and GenScript Inc. (Nanjing, China). .. Using a luciferase-expressing lentiviral pseudotype system, we expressed WT and N501Y.V1 (variant 1) and N501Y.V2 (variant 2) mutant Spike proteins in enveloped virions.

    Clone Assay:

    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity
    Article Snippet: Our study will be helpful for understanding SARS-CoV-2 transmission and for the design of vaccines and therapeutic interventions against COVID-19. .. Plasmids The codon-optimized gene encoding SARS-CoV-2 S protein (GenBank: QHD43416) with C-terminal 19-amino acid deletion was synthesized by Sino Biological Inc (Beijing, China), and cloned into the Kpn I and Xba I restriction sites of pCMV3 vector (pCMV3-SARS-CoV-2-S-C19del, denoted as pS-D614). .. The D614G mutant S-expressing plasmid (denoted as pS-G614) was constructed by site-directed mutagenesis, with pS-D614 plasmid as a template.

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and was used interchangeably. .. To construct a replication-competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 S protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene; 1786) or a catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

    Plasmid Preparation:

    Article Title: D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity
    Article Snippet: Our study will be helpful for understanding SARS-CoV-2 transmission and for the design of vaccines and therapeutic interventions against COVID-19. .. Plasmids The codon-optimized gene encoding SARS-CoV-2 S protein (GenBank: QHD43416) with C-terminal 19-amino acid deletion was synthesized by Sino Biological Inc (Beijing, China), and cloned into the Kpn I and Xba I restriction sites of pCMV3 vector (pCMV3-SARS-CoV-2-S-C19del, denoted as pS-D614). .. The D614G mutant S-expressing plasmid (denoted as pS-G614) was constructed by site-directed mutagenesis, with pS-D614 plasmid as a template.

    Article Title: Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2
    Article Snippet: .. Generation of deletion mutants of the SARS-CoV-2 S proteinThe codon-optimized version of the open reading frame cDNA for SARS-CoV-2 S protein was purchased from Sino Biological in the vector pCMV3-SP-N-Myc. .. The deletion mutants of the SARS-CoV-2 S were generated by the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Cat.200522) according to manufacturer’s instructions.

    Expressing:

    Article Title: A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine
    Article Snippet: All viral stocks were analyzed by deep sequencing after propagation to verify the integrity of the original viral genome. .. Lentivirus pseudotyped virusCloning of expression constructs: For generation of spike protein pseudotyped lentivirus (Lenti-CoV2), a codon optimized SARS-CoV-2 spike protein (accession #QHD43416.1) construct was obtained from Sino Biologicals. ..

    Construct:

    Article Title: A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine
    Article Snippet: All viral stocks were analyzed by deep sequencing after propagation to verify the integrity of the original viral genome. .. Lentivirus pseudotyped virusCloning of expression constructs: For generation of spike protein pseudotyped lentivirus (Lenti-CoV2), a codon optimized SARS-CoV-2 spike protein (accession #QHD43416.1) construct was obtained from Sino Biologicals. ..

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and was used interchangeably. .. To construct a replication-competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 S protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene; 1786) or a catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

    Mutagenesis:

    Article Title: Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies
    Article Snippet: .. DNA sequences encoding the reference strain wild-type (WT) and mutant Spike proteins of SARS-CoV-2 were codon-optimized and synthesized by Sino Biological Inc. (Beijing, China) and GenScript Inc. (Nanjing, China). .. Using a luciferase-expressing lentiviral pseudotype system, we expressed WT and N501Y.V1 (variant 1) and N501Y.V2 (variant 2) mutant Spike proteins in enveloped virions.

    Sequencing:

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and was used interchangeably. .. To construct a replication-competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 S protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene; 1786) or a catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

    Recombinant:

    Article Title: Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses
    Article Snippet: A second construct derived from a codon-optimized plasmid (SinoBiological) behaved identically in our assays and was used interchangeably. .. To construct a replication-competent rVSV/SARS-CoV-2 chimeric virus clone, a codon-optimized cDNA sequence encoding the SARS-CoV-2 S protein (SinoBiological) but lacking the C-terminal 18 codons was inserted, using Gibson cloning, into a recombinant VSV background that contains GFP immediately upstream of the L (polymerase) following a strategy we previously described for the exchange of VSV-G with HIV-1 Env proteins ( ). .. An ACE2 lentivirus expression CS(ACE2)IB vector was constructed by inserting a cDNA encoding an unaltered ACE2 (Addgene; 1786) or a catalytically inactive ACE2 mutant (ACE2-H374N & H378N) into the lentivirus expression vector CSIB ( ).

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  • 94
    Sino Biological soluble cd38
    <t>CD38</t> and GDF15 expression and circulating levels are increased in AAA patients. ( A ) Human abdominal aortic mRNA levels of CD38 measured by quantitative real-time PCR and normalized to β-actin in healthy donors ( n = 15) and patients (AAA) ( n = 80). ( B ) CD38 plasma levels in AAA ( n = 94) vs. healthy donors ( n = 46); ( C ) plasma levels of GDF15 in AAA ( n = 94) vs. healthy donors ( n = 46). ( D ) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 ( n = 10; scale bars: 100 µm). ( E ) Representative images of immunostaining assays performed in abdominal aorta sections targeting GDF15 ( n = 0; scale bars: 100 µm). AAA (-) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells.
    Soluble Cd38, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sino Biological human vwf elisa kit
    DIDS inhibits stimulus induced <t>vWF</t> release from normoxic HUVECs. The Ca 2+ ionophore A23187 induces vWF extrusion from HUVECs in a non-pathological model of vesicular release. DIDS abolishes stimulus-evoked vWF release. ( A ) Confocal Z-stack projection fluorescent images of vWF localization (red) in HUVECs treated as indicated. Arrows indicate vWF released extracellularly. ( B ) Summary of supernatant vWF expression measured by <t>ELISA.</t> Data are mean ±SEM from 3 separate 15-min experiments. Asterisks (*) indicate significant difference from normoxic controls; black bars indicate significance between connected treatments ( p
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    93
    Sino Biological hiv 1 infection
    NSC- and A549-derived exosomes significantly enhance <t>HIV-1</t> entry into human immune cell lines. Notes: ( A , C ) YU-2 virus entry into A3R5.7 cells was evaluated in the presence or absence of ( A ) NSC-derived exosomes (0.1 μg) or ( C ) A549-dervied exosomes (0.1 μg). ( B , D ) The differentiated THP2574 cell line was used for entry experiments with YU-2 in the presence or absence of ( B ) NSC-derived exosomes (0.1 μg) or ( D ) A549-derived exosomes (0.1 μg). Virus entry was also evaluated in the presence of exosomes and anti-TIM-4 antibody. Viral gene expression in all control and treatment groups was assessed by Renilla luciferase activity at 72 h post-infection. Data represent 12 independent experiments. Significant differences between treatment groups were determined by one-way ANOVA * P
    Hiv 1 Infection, 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
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    Sino Biological human tnfr1
    EV‐NID1 and serum <t>TNFR1</t> levels correlate with the tumor stage of HCC. A) ELISA analysis of NID1 expression in circulating EVs obtained from sera collected from individuals without liver disease (Control) ( n = 12), patients with early ( n = 43) and late stage ( n = 22) HCC (left). ELISA analysis of serum TNFR1 in the same subjects (right). ELISA was performed in duplicate. B) Correlation between EV‐NID1 and serum TNFR1 levels determined in (A) using Pearson correlation test. C) ROC curves of EV‐NID1, serum TNFR1, and combined EV‐NID1 and serum TNFR1 for discriminating control subjects and patients with early stage HCC. D) ROC curves of AFP, AFP in combination with EV‐NID1, or serum TNFR1 for discriminating control subjects and patients with early stage HCC. E) Proposed signaling mediated by EV‐NID1. The EV‐NID1 level increases with HCC development. EV‐NID1 derived from metastatic HCC cells promotes liver tumor development and distant metastasis to the lungs. EV‐NID1 increases pulmonary vessel leakiness, angiogenesis, and colonization of cancer cells to the lungs and activates pulmonary fibroblasts to secrete TNFR1, which in turn promotes HCC cell growth and motility. ROC, receiver operating characteristic. Data are represented as the mean ± SEM; : p
    Human Tnfr1, 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
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    CD38 and GDF15 expression and circulating levels are increased in AAA patients. ( A ) Human abdominal aortic mRNA levels of CD38 measured by quantitative real-time PCR and normalized to β-actin in healthy donors ( n = 15) and patients (AAA) ( n = 80). ( B ) CD38 plasma levels in AAA ( n = 94) vs. healthy donors ( n = 46); ( C ) plasma levels of GDF15 in AAA ( n = 94) vs. healthy donors ( n = 46). ( D ) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 ( n = 10; scale bars: 100 µm). ( E ) Representative images of immunostaining assays performed in abdominal aorta sections targeting GDF15 ( n = 0; scale bars: 100 µm). AAA (-) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells.

    Journal: Antioxidants

    Article Title: Oxidative Stress and Inflammatory Markers in Abdominal Aortic Aneurysm

    doi: 10.3390/antiox10040602

    Figure Lengend Snippet: CD38 and GDF15 expression and circulating levels are increased in AAA patients. ( A ) Human abdominal aortic mRNA levels of CD38 measured by quantitative real-time PCR and normalized to β-actin in healthy donors ( n = 15) and patients (AAA) ( n = 80). ( B ) CD38 plasma levels in AAA ( n = 94) vs. healthy donors ( n = 46); ( C ) plasma levels of GDF15 in AAA ( n = 94) vs. healthy donors ( n = 46). ( D ) Representative images of immunostaining assays performed in abdominal aorta sections from donors and AAA patients targeting CD38 ( n = 10; scale bars: 100 µm). ( E ) Representative images of immunostaining assays performed in abdominal aorta sections targeting GDF15 ( n = 0; scale bars: 100 µm). AAA (-) indicates negative control for immunohistochemistry. Arrows indicate the positively stained cells.

    Article Snippet: ELISA KitsThe circulating levels of soluble IgM (ab214568, Abcam, UK), IgG (ab195215), soluble CD38 (KIT10818-1, Sino Biological Inc., Wayne, PA, USA), soluble CD36 (ABE-196-02, Nordic BioSite, Täby, Sweden), S100A4 (CSB-EL020632HU, Cusabio Biotech Co, LTD, Beijing, China) and GDF15 (Quantikine ELISA Human GDF15, DGD150; R & D Systems, Minneapolis, MN, USA), in plasma from patients were measured using commercially available ELISA kits in accordance with the manufacturer’s instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Negative Control, Immunohistochemistry, Staining

    IgG, CD38 and GDF15 circulating levels positively correlate with the AAA diameter whereas only CD38 correlates with PWS. ( A ) Graphs showing the correlation analysis between IgG plasma levels and AAA diameter ( n = 90) and ( B , C ) the correlation analysis between CD38 plasma levels and AAA diameter ( n = 90) or PWS values in AAA patients ( n = 58). ( D ) Graph showing the correlation analysis between GDF15 plasma levels and AAA diameter ( n = 90). The r and p -values are obtained by performing the Spearman or the Pearson correlation coefficient test. Results are expressed as mean ± SEM.

    Journal: Antioxidants

    Article Title: Oxidative Stress and Inflammatory Markers in Abdominal Aortic Aneurysm

    doi: 10.3390/antiox10040602

    Figure Lengend Snippet: IgG, CD38 and GDF15 circulating levels positively correlate with the AAA diameter whereas only CD38 correlates with PWS. ( A ) Graphs showing the correlation analysis between IgG plasma levels and AAA diameter ( n = 90) and ( B , C ) the correlation analysis between CD38 plasma levels and AAA diameter ( n = 90) or PWS values in AAA patients ( n = 58). ( D ) Graph showing the correlation analysis between GDF15 plasma levels and AAA diameter ( n = 90). The r and p -values are obtained by performing the Spearman or the Pearson correlation coefficient test. Results are expressed as mean ± SEM.

    Article Snippet: ELISA KitsThe circulating levels of soluble IgM (ab214568, Abcam, UK), IgG (ab195215), soluble CD38 (KIT10818-1, Sino Biological Inc., Wayne, PA, USA), soluble CD36 (ABE-196-02, Nordic BioSite, Täby, Sweden), S100A4 (CSB-EL020632HU, Cusabio Biotech Co, LTD, Beijing, China) and GDF15 (Quantikine ELISA Human GDF15, DGD150; R & D Systems, Minneapolis, MN, USA), in plasma from patients were measured using commercially available ELISA kits in accordance with the manufacturer’s instructions.

    Techniques:

    DIDS inhibits stimulus induced vWF release from normoxic HUVECs. The Ca 2+ ionophore A23187 induces vWF extrusion from HUVECs in a non-pathological model of vesicular release. DIDS abolishes stimulus-evoked vWF release. ( A ) Confocal Z-stack projection fluorescent images of vWF localization (red) in HUVECs treated as indicated. Arrows indicate vWF released extracellularly. ( B ) Summary of supernatant vWF expression measured by ELISA. Data are mean ±SEM from 3 separate 15-min experiments. Asterisks (*) indicate significant difference from normoxic controls; black bars indicate significance between connected treatments ( p

    Journal: PLoS ONE

    Article Title: DIDS Prevents Ischemic Membrane Degradation in Cultured Hippocampal Neurons by Inhibiting Matrix Metalloproteinase Release

    doi: 10.1371/journal.pone.0043995

    Figure Lengend Snippet: DIDS inhibits stimulus induced vWF release from normoxic HUVECs. The Ca 2+ ionophore A23187 induces vWF extrusion from HUVECs in a non-pathological model of vesicular release. DIDS abolishes stimulus-evoked vWF release. ( A ) Confocal Z-stack projection fluorescent images of vWF localization (red) in HUVECs treated as indicated. Arrows indicate vWF released extracellularly. ( B ) Summary of supernatant vWF expression measured by ELISA. Data are mean ±SEM from 3 separate 15-min experiments. Asterisks (*) indicate significant difference from normoxic controls; black bars indicate significance between connected treatments ( p

    Article Snippet: ELISA HUVEC vWF release was quantified using a human vWF ELISA kit according to the manufacturer's protocol (Sino Biological Inc., Beijing, CH), using mouse anti-vWF monoclonal antibody and biotinylated rabbit anti-vWF polyclonal antibody as the capture and detection antibodies, respectively.

    Techniques: Expressing, Enzyme-linked Immunosorbent Assay

    NSC- and A549-derived exosomes significantly enhance HIV-1 entry into human immune cell lines. Notes: ( A , C ) YU-2 virus entry into A3R5.7 cells was evaluated in the presence or absence of ( A ) NSC-derived exosomes (0.1 μg) or ( C ) A549-dervied exosomes (0.1 μg). ( B , D ) The differentiated THP2574 cell line was used for entry experiments with YU-2 in the presence or absence of ( B ) NSC-derived exosomes (0.1 μg) or ( D ) A549-derived exosomes (0.1 μg). Virus entry was also evaluated in the presence of exosomes and anti-TIM-4 antibody. Viral gene expression in all control and treatment groups was assessed by Renilla luciferase activity at 72 h post-infection. Data represent 12 independent experiments. Significant differences between treatment groups were determined by one-way ANOVA * P

    Journal: International Journal of Nanomedicine

    Article Title: Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells

    doi: 10.2147/IJN.S132762

    Figure Lengend Snippet: NSC- and A549-derived exosomes significantly enhance HIV-1 entry into human immune cell lines. Notes: ( A , C ) YU-2 virus entry into A3R5.7 cells was evaluated in the presence or absence of ( A ) NSC-derived exosomes (0.1 μg) or ( C ) A549-dervied exosomes (0.1 μg). ( B , D ) The differentiated THP2574 cell line was used for entry experiments with YU-2 in the presence or absence of ( B ) NSC-derived exosomes (0.1 μg) or ( D ) A549-derived exosomes (0.1 μg). Virus entry was also evaluated in the presence of exosomes and anti-TIM-4 antibody. Viral gene expression in all control and treatment groups was assessed by Renilla luciferase activity at 72 h post-infection. Data represent 12 independent experiments. Significant differences between treatment groups were determined by one-way ANOVA * P

    Article Snippet: Blocking of HIV-1 infection A protocol similar to HIV-1 infection was performed but with addition of 0.2 μg/well anti-mouse TIM-4 or anti-human TIM-4 (Sino Biological, Inc.) to the YU-2/exosome/cell incubation or with YU-2 only as a control.

    Techniques: Derivative Assay, Expressing, Luciferase, Activity Assay, Infection

    Breast milk- and plasma-derived exosomes enhance HIV-1 entry into human immune cell lines. Notes: ( A , C ) YU-2 virus entry into A3R5.7 cells was evaluated in the presence or absence of ( A ) breast milk-derived exosomes (0.035 μg) or ( C ) plasma-derived exosomes (0.05 μg). Virus entry was evaluated in the presence of exosomes and anti-TIM-4 antibodies. ( B , D ) The differentiated THP2574 cell line was used for entry experiments with YU-2 in the presence or absence of ( B ) breast milk-derived exosomes (0.035 μg) or ( D ) plasma-derived exosomes (0.05 μg). Virus entry was also evaluated in the presence of exosomes and anti-TIM-4 antibody. Viral gene expression in all control and treatment groups was assessed by Renilla luciferase activity at 72 h post-infection. Data represent 12 independent experiments. Significant differences between treatment groups were determined by one-way ANOVA. ** P

    Journal: International Journal of Nanomedicine

    Article Title: Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells

    doi: 10.2147/IJN.S132762

    Figure Lengend Snippet: Breast milk- and plasma-derived exosomes enhance HIV-1 entry into human immune cell lines. Notes: ( A , C ) YU-2 virus entry into A3R5.7 cells was evaluated in the presence or absence of ( A ) breast milk-derived exosomes (0.035 μg) or ( C ) plasma-derived exosomes (0.05 μg). Virus entry was evaluated in the presence of exosomes and anti-TIM-4 antibodies. ( B , D ) The differentiated THP2574 cell line was used for entry experiments with YU-2 in the presence or absence of ( B ) breast milk-derived exosomes (0.035 μg) or ( D ) plasma-derived exosomes (0.05 μg). Virus entry was also evaluated in the presence of exosomes and anti-TIM-4 antibody. Viral gene expression in all control and treatment groups was assessed by Renilla luciferase activity at 72 h post-infection. Data represent 12 independent experiments. Significant differences between treatment groups were determined by one-way ANOVA. ** P

    Article Snippet: Blocking of HIV-1 infection A protocol similar to HIV-1 infection was performed but with addition of 0.2 μg/well anti-mouse TIM-4 or anti-human TIM-4 (Sino Biological, Inc.) to the YU-2/exosome/cell incubation or with YU-2 only as a control.

    Techniques: Derivative Assay, Expressing, Luciferase, Activity Assay, Infection

    EV‐NID1 and serum TNFR1 levels correlate with the tumor stage of HCC. A) ELISA analysis of NID1 expression in circulating EVs obtained from sera collected from individuals without liver disease (Control) ( n = 12), patients with early ( n = 43) and late stage ( n = 22) HCC (left). ELISA analysis of serum TNFR1 in the same subjects (right). ELISA was performed in duplicate. B) Correlation between EV‐NID1 and serum TNFR1 levels determined in (A) using Pearson correlation test. C) ROC curves of EV‐NID1, serum TNFR1, and combined EV‐NID1 and serum TNFR1 for discriminating control subjects and patients with early stage HCC. D) ROC curves of AFP, AFP in combination with EV‐NID1, or serum TNFR1 for discriminating control subjects and patients with early stage HCC. E) Proposed signaling mediated by EV‐NID1. The EV‐NID1 level increases with HCC development. EV‐NID1 derived from metastatic HCC cells promotes liver tumor development and distant metastasis to the lungs. EV‐NID1 increases pulmonary vessel leakiness, angiogenesis, and colonization of cancer cells to the lungs and activates pulmonary fibroblasts to secrete TNFR1, which in turn promotes HCC cell growth and motility. ROC, receiver operating characteristic. Data are represented as the mean ± SEM; : p

    Journal: Advanced Science

    Article Title: Nidogen 1‐Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1, Nidogen 1‐Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1

    doi: 10.1002/advs.202002157

    Figure Lengend Snippet: EV‐NID1 and serum TNFR1 levels correlate with the tumor stage of HCC. A) ELISA analysis of NID1 expression in circulating EVs obtained from sera collected from individuals without liver disease (Control) ( n = 12), patients with early ( n = 43) and late stage ( n = 22) HCC (left). ELISA analysis of serum TNFR1 in the same subjects (right). ELISA was performed in duplicate. B) Correlation between EV‐NID1 and serum TNFR1 levels determined in (A) using Pearson correlation test. C) ROC curves of EV‐NID1, serum TNFR1, and combined EV‐NID1 and serum TNFR1 for discriminating control subjects and patients with early stage HCC. D) ROC curves of AFP, AFP in combination with EV‐NID1, or serum TNFR1 for discriminating control subjects and patients with early stage HCC. E) Proposed signaling mediated by EV‐NID1. The EV‐NID1 level increases with HCC development. EV‐NID1 derived from metastatic HCC cells promotes liver tumor development and distant metastasis to the lungs. EV‐NID1 increases pulmonary vessel leakiness, angiogenesis, and colonization of cancer cells to the lungs and activates pulmonary fibroblasts to secrete TNFR1, which in turn promotes HCC cell growth and motility. ROC, receiver operating characteristic. Data are represented as the mean ± SEM; : p

    Article Snippet: Human AFP ELISA Kit (Solarbio) and Human TNFR1 (Sino Biological) were used to determine the level of AFP and TNFR1 in serum of patients.

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay

    TNFR1 secreted by EV‐NID1‐activated pulmonary fibroblasts promotes HCC cell motility and colonization in the lungs. A) Immunohistochemistry of metastatic lesions in lungs tissues obtained from mice injected with PBS, MHCC97L CTL‐KD‐EVs of NID1‐KD‐EVs using anti‐ α ‐SMA antibody. Magnification, 20 ×; Scale bar, 25 µm. B) Immunoblotting of S100A4 expression in MRC‐5 cells treated with EVs for 24 h. C) Immunofluorescence in MRC‐5 cells after a 24 h incubation with PKH67‐labeled MHCC97L‐EVs. Scale bar: 20 µm. D) Analysis of TNFR1 copy number and concentration of soluble TNFR1 in MRC‐5 cells pretreated with the indicated EVs using qPCR and ELISA, respectively. E) Diagram illustrating the collection of conditioned medium from MRC‐5 cells pretreated with EVs for functional assays. F) Colony formation assay performed with Hep3B incubated with the conditioned medium from MRC‐5 cells incubation with EVs from CTL‐KD or NID1‐KD cells for 72 h. Anti‐TNFR1 neutralizing antibody was added to neutralize the activity of soluble TNFR1 (Ab) (0.4 µg mL −1 ) in the conditioned medium. Representative image shows the fixed and crystal violet‐stained colonies. G) Migration and invasion assays performed using PLC/PRF/5 cells pretreated as described in (F). Representative image shows the fixed and crystal violet‐stained migratory and invasive cells. H) Bioluminescence imaging of mice ( n = 6) subjected to intravenous coinjection of murine p53−/−; Myc hepatoblasts (1 × 10 5 ) with PBS, IgG (10 µg), or anti‐TNFR1 antibody (TNFR1 Ab) (10 µg). Quantification of the luciferase signal is shown. I) Ex vivo bioluminescence imaging of lung tissues. Quantification of the luciferase signal is shown. J) Representative images of H E staining of lung tissues. Examples of metastatic lesions are indicated by arrowheads. Insets show the enlarged area of the metastatic lesions. Magnification, 2.5 ×; Scale bar, 500 µm. Three independent experiments were performed in triplicate for assays shown in (D)–(G). Data are represented as the mean ± SEM; : p

    Journal: Advanced Science

    Article Title: Nidogen 1‐Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1, Nidogen 1‐Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1

    doi: 10.1002/advs.202002157

    Figure Lengend Snippet: TNFR1 secreted by EV‐NID1‐activated pulmonary fibroblasts promotes HCC cell motility and colonization in the lungs. A) Immunohistochemistry of metastatic lesions in lungs tissues obtained from mice injected with PBS, MHCC97L CTL‐KD‐EVs of NID1‐KD‐EVs using anti‐ α ‐SMA antibody. Magnification, 20 ×; Scale bar, 25 µm. B) Immunoblotting of S100A4 expression in MRC‐5 cells treated with EVs for 24 h. C) Immunofluorescence in MRC‐5 cells after a 24 h incubation with PKH67‐labeled MHCC97L‐EVs. Scale bar: 20 µm. D) Analysis of TNFR1 copy number and concentration of soluble TNFR1 in MRC‐5 cells pretreated with the indicated EVs using qPCR and ELISA, respectively. E) Diagram illustrating the collection of conditioned medium from MRC‐5 cells pretreated with EVs for functional assays. F) Colony formation assay performed with Hep3B incubated with the conditioned medium from MRC‐5 cells incubation with EVs from CTL‐KD or NID1‐KD cells for 72 h. Anti‐TNFR1 neutralizing antibody was added to neutralize the activity of soluble TNFR1 (Ab) (0.4 µg mL −1 ) in the conditioned medium. Representative image shows the fixed and crystal violet‐stained colonies. G) Migration and invasion assays performed using PLC/PRF/5 cells pretreated as described in (F). Representative image shows the fixed and crystal violet‐stained migratory and invasive cells. H) Bioluminescence imaging of mice ( n = 6) subjected to intravenous coinjection of murine p53−/−; Myc hepatoblasts (1 × 10 5 ) with PBS, IgG (10 µg), or anti‐TNFR1 antibody (TNFR1 Ab) (10 µg). Quantification of the luciferase signal is shown. I) Ex vivo bioluminescence imaging of lung tissues. Quantification of the luciferase signal is shown. J) Representative images of H E staining of lung tissues. Examples of metastatic lesions are indicated by arrowheads. Insets show the enlarged area of the metastatic lesions. Magnification, 2.5 ×; Scale bar, 500 µm. Three independent experiments were performed in triplicate for assays shown in (D)–(G). Data are represented as the mean ± SEM; : p

    Article Snippet: Human AFP ELISA Kit (Solarbio) and Human TNFR1 (Sino Biological) were used to determine the level of AFP and TNFR1 in serum of patients.

    Techniques: Immunohistochemistry, Mouse Assay, Injection, Expressing, Immunofluorescence, Incubation, Labeling, Concentration Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Functional Assay, Colony Assay, Activity Assay, Staining, Migration, Planar Chromatography, Imaging, Luciferase, Ex Vivo

    Treatment with TNFR1 neutralizing antibody effectively inhibits tumor growth and metastasis in mice implanted with metastatic tumor seed. A) Schematic diagram of the treatment regimen applied to mice implanted with luciferase‐labeled MHCC97L cells in the liver. Mice were administered PBS, IgG, or anti‐TNFR1 antibody (200 µg) via peritoneal injection every 4 days for 28 days ( n = 5). B) Bioluminescence imaging of animals at the end of the experiment. Quantification of the luciferase signal is shown. The size of the liver tumors was measured and plotted. C) Ex vivo bioluminescence imaging of livers. Quantification of the luciferase signal is shown. D) Representative image of H E staining of liver tissues showing the boundary of tumors obtained from (C). Dotted line indicates the bulging growth fronts of liver tumor. Arrows indicate the cluster of tumors nearby the liver‐tumor boundary. Magnification, 20 ×; Scale bar, 100 nm. E) Ex vivo bioluminescence imaging of lungs. Quantification of the luciferase signal is shown. F) Body weight of the mice was measured twice a week and plotted against time. Data are represented as the mean ± SEM; : p

    Journal: Advanced Science

    Article Title: Nidogen 1‐Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1, Nidogen 1‐Enriched Extracellular Vesicles Facilitate Extrahepatic Metastasis of Liver Cancer by Activating Pulmonary Fibroblasts to Secrete Tumor Necrosis Factor Receptor 1

    doi: 10.1002/advs.202002157

    Figure Lengend Snippet: Treatment with TNFR1 neutralizing antibody effectively inhibits tumor growth and metastasis in mice implanted with metastatic tumor seed. A) Schematic diagram of the treatment regimen applied to mice implanted with luciferase‐labeled MHCC97L cells in the liver. Mice were administered PBS, IgG, or anti‐TNFR1 antibody (200 µg) via peritoneal injection every 4 days for 28 days ( n = 5). B) Bioluminescence imaging of animals at the end of the experiment. Quantification of the luciferase signal is shown. The size of the liver tumors was measured and plotted. C) Ex vivo bioluminescence imaging of livers. Quantification of the luciferase signal is shown. D) Representative image of H E staining of liver tissues showing the boundary of tumors obtained from (C). Dotted line indicates the bulging growth fronts of liver tumor. Arrows indicate the cluster of tumors nearby the liver‐tumor boundary. Magnification, 20 ×; Scale bar, 100 nm. E) Ex vivo bioluminescence imaging of lungs. Quantification of the luciferase signal is shown. F) Body weight of the mice was measured twice a week and plotted against time. Data are represented as the mean ± SEM; : p

    Article Snippet: Human AFP ELISA Kit (Solarbio) and Human TNFR1 (Sino Biological) were used to determine the level of AFP and TNFR1 in serum of patients.

    Techniques: Mouse Assay, Luciferase, Labeling, Injection, Imaging, Ex Vivo, Staining