Journal: Respiratory Research

Article Title: IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β 2 integrin and CXCR3

doi: 10.1186/1465-9921-12-138

Figure Lengend Snippet: Anti-β 2 integrin mAb and anti-CXCR3 mAb suppress eosinophil adhesion enhanced by ICAM-1 and IP-10 . (A) IP-10-enhanced eosinophil adhesion to ICAM-1 is inhibited by anti-β 2 integrin mAb. Eosinophils were pre-incubated with either anti-integrin mAb (3 μg/ml) or control IgG for 15 min prior to analysis of adhesion in the presence or absence of IP-10. Data are shown as means ± SEM of 6 experiments using cells from different donors. (B) IP-10-enhanced eosinophil adhesion to ICAM-1 is inhibited by anti-CXCR3 mAb. Eosinophils were pre-incubated with either anti-CXCR3 mAb (3 μg/ml) or control IgG for 15 min prior to analysis of adhesion in the presence or absence of IP-10. Data are shown as means ± SEM of six experiments using cells from different donors. Open and filled bars, eosinophil adhesion without and with IP-10, respectively. ** P < 0.01 versus without IP-10. ## P < 0.01, ### P < 0.001 versus mouse IgG1.

Article Snippet: In selected experiments, eosinophils were incubated with anti-α 4 -integrin mAb (3 μg/ml; clone HP1/2; Cosmo Bio Co. Ltd, Tokyo, Japan), anti-β 2 -integrin mAb (3 μg/ml; clone L130; Becton Dickinson, Franklin Lakes, NJ), or anti-CXCR3 mAb (3 μg/ml; clone 49801; R&D Systems, Minneapolis, MN) before assay.

Techniques: Incubation, Control

Journal: Respiratory Research

Article Title: IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β 2 integrin and CXCR3

doi: 10.1186/1465-9921-12-138

Figure Lengend Snippet: IP-10 significantly enhances eosinophil O 2 - generation . (A) IP-10 significantly enhances eosinophil O 2 - generation in the presence of ICAM-1. Eosinophil cell density was adjusted to 1.25 × 10 6 cells/ml of HBSS/gel mixed 4:1 with cytochrome C, and the eosinophil suspension was then added to ICAM-1 coated 96-well plates. Immediately after adding IP-10 (30 or 100 nM), eosinophil O 2 - generation was measured based on the SOD-inhibitable reduction of cytochrome C. Data are shown as means ± SEM of 5 experiments using cells from different donors. (B) Eosinophil O 2 - generation induced by the combination of IP-10 + ICAM-1 is blocked by anti-β 2 integrin mAb. Eosinophils were pre-incubated with anti-β 2 integrin mAb (3 μg/ml) or control IgG for 15 min prior to analysis of O 2 - generation of eosinophils as in (A). Data are shown as means ± SEM of 7 experiments using cells from different donors. (C) Eosinophil O 2 - generation induced by a combination of IP-10 + ICAM-1 is blocked by anti-CXCR3 mAb. Eosinophils were pre-incubated with anti-CXCR3 mAb (3 μg/ml) or control IgG for 15 min prior to analysis of O 2 - generation of eosinophils as in (A). Data are shown as means ± SEM of 7 experiments using cells from different donors. Open and filled bars, eosinophil O 2 - generation without and with IP-10, respectively. * P < 0.05 versus without IP-10. # P < 0.05 versus mouse IgG1.

Article Snippet: In selected experiments, eosinophils were incubated with anti-α 4 -integrin mAb (3 μg/ml; clone HP1/2; Cosmo Bio Co. Ltd, Tokyo, Japan), anti-β 2 -integrin mAb (3 μg/ml; clone L130; Becton Dickinson, Franklin Lakes, NJ), or anti-CXCR3 mAb (3 μg/ml; clone 49801; R&D Systems, Minneapolis, MN) before assay.

Techniques: Suspension, Incubation, Control

Journal: Respiratory Research

Article Title: IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β 2 integrin and CXCR3

doi: 10.1186/1465-9921-12-138

Figure Lengend Snippet: Other CXCR3 ligands also enhance eosinophil adhesion and eosinophil O 2 - generation . (A-C) All tested CXCR3 ligands significantly augmented eosinophil adhesion to ICAM-1-coated plates. Eosinophils were incubated with or without CXCR3 ligands for 20 min, and adhesion of eosinophils was then examined. (A) All of the CXCR3 ligands (100 nM) augmented eosinophil adhesion to ICAM-1. (B) Dose-response relationship between Mig and eosinophil adhesion. (C) Dose-response relationship between I-TAC and eosinophil adhesion. Data shown are means ± SEM of 5 experiments using cells from different donors. (D-F) All tested CXCR3 ligands significantly induced eosinophil O 2 - generation in the presence of ICAM-1. Eosinophil O 2 - generation was measured with or without CXCR3 ligands. (D) All of the CXCR3 ligands (100 nM) induced eosinophil O 2 - generation. (E) Dose-response relationship between Mig and eosinophil O 2 - generation. (F) Dose-response relationship between I-TAC and eosinophil O 2 - generation. Data shown are means ± SEM of 5 experiments using cells from different donors. * P < 0.05, ** P < 0.01 versus without CXCR3 ligands.

Article Snippet: In selected experiments, eosinophils were incubated with anti-α 4 -integrin mAb (3 μg/ml; clone HP1/2; Cosmo Bio Co. Ltd, Tokyo, Japan), anti-β 2 -integrin mAb (3 μg/ml; clone L130; Becton Dickinson, Franklin Lakes, NJ), or anti-CXCR3 mAb (3 μg/ml; clone 49801; R&D Systems, Minneapolis, MN) before assay.

Techniques: Incubation

Journal: Respiratory Research

Article Title: IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β 2 integrin and CXCR3

doi: 10.1186/1465-9921-12-138

Figure Lengend Snippet: IP-10, but not Mig or I-TAC, induces eosinophil release of EDN . Eosinophils (1 × 10 6 cells/ml) in 96-well plates were incubated for the 240-min required to measure O 2 - generation, immediately centrifuged and the concentration of EDN in cell-free supernatants was then quantified using ELISA. Data shown are means ± SEM of 6 experiments using cells from different donors. * P < 0.05 versus without CXCR3 ligands.

Article Snippet: In selected experiments, eosinophils were incubated with anti-α 4 -integrin mAb (3 μg/ml; clone HP1/2; Cosmo Bio Co. Ltd, Tokyo, Japan), anti-β 2 -integrin mAb (3 μg/ml; clone L130; Becton Dickinson, Franklin Lakes, NJ), or anti-CXCR3 mAb (3 μg/ml; clone 49801; R&D Systems, Minneapolis, MN) before assay.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Respiratory Research

Article Title: IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β 2 integrin and CXCR3

doi: 10.1186/1465-9921-12-138

Figure Lengend Snippet: Production of cytokines and chemokines by eosinophils stimulated with CXCR3 ligands.

Article Snippet: In selected experiments, eosinophils were incubated with anti-α 4 -integrin mAb (3 μg/ml; clone HP1/2; Cosmo Bio Co. Ltd, Tokyo, Japan), anti-β 2 -integrin mAb (3 μg/ml; clone L130; Becton Dickinson, Franklin Lakes, NJ), or anti-CXCR3 mAb (3 μg/ml; clone 49801; R&D Systems, Minneapolis, MN) before assay.

Techniques: Control

Journal: Gene therapy

Article Title: Therapeutic effect of CXCR3-expressing regulatory T cells on liver, lung and intestinal damages in a murine acute GVHD model.

doi: 10.1038/sj.gt.3303051

Figure Lengend Snippet: Figure 4 Histopathological graft-vs-host disease (GVHD) evalua- tion of target organs in nTreg cell-, Treg cell- and CXCR3-Treg cell- transferred recipient mice. Histopathological analysis of the tissue sections was performed at 4 weeks after injection of regulatory T cells in all groups. The severity of GVHD was assessed by histopathological analysis of liver, intestine and lung using a semiquantitative scoring system as described in the ‘Materials and methods’ section. Nontransferred GVHD-induced B6D2F1 recipient mice were used as control. Values are the mean and SD (n ¼ 6–8 mice per group). Similar results were observed in two independent experiments. Statistical analysis was performed using Student’s t-test (*Po0.05 vs control. **Po0.05 vs 4 105 Treg. ***Po0.05 vs 1 106 Treg).

Article Snippet: Mouse CXCL10, IL-2 and the rat anti-mouse CXCR3 monoclonal antibody (MoAb) (220803) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Injection, Control

Journal: Journal of Biological Chemistry

Article Title: Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation

doi: 10.1074/jbc.m300470200

Figure Lengend Snippet: FIG. 1. Preparation of recombinant caprine IP-10 and the antibody. A, re- combinant caprine IP-10 (rcIP-10) was ex- pressed in E. coli BL21-SI, and cell ly- sates before (lane 1) and after (lane 2) purification using a nickel-chelating col- umn was subjected to SDS-PAGE. B, pu- rified rcIP-10 protein (50 or 200 ng/lane) was subjected to Western blot analysis with either anti-histidine tag (lane 1) or anti-caprine IP-10 antibody (30 g/ml) generated in our laboratory (lane 2). C, left, Northern blot analysis of CXCR3 mRNA. RNA was extracted from KU-1 cells that had been transiently trans- fected with pcDNA3.1-caprine CXCR3 (CXCR3) or parental pcDNA3.1 (Mock). Right, biological activity of rcIP-10 to CXCR3 transfected KU-1 cells expressing (circle) or not expressing (square) CXCR3 was tested by chemotaxis assay. Bars rep- resent LSM S.E. D, chemotaxis activity was demonstrated in KU-1 cells with the addition of 20 ng/ml rcIP-10, to which no further treatment was applied (), or neutralized by the pretreatment with the anti-caprine IP-10 antibody (anti IP-10, 30 g/ml), or with control rabbit IgG (con- trol IgG). An asterisk indicates a signifi- cant difference (p 0.05). For the results shown in C, right panel, and D, triplicate samples were examined for each treat- ment, and three independent experi- ments were performed.

Article Snippet: Nonspecific binding was blocked by a treatment with Block Ace at room temperature for 1 h, and slides were then incubated with a mouse monoclonal antibody to human CXCR3 (10 g/ml, R&D Systems Inc., Minneapolis, MN) or normal mouse IgG (Sigma) at 4 °C for 12 h. After the primary antibody incubation, slides were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (15 g/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. Nuclei were stained with propidium iodide (2 g/ml, Sigma).

Techniques: Recombinant, Purification, SDS Page, Western Blot, Generated, Northern Blot, Activity Assay, Transfection, Expressing, Chemotaxis Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation

doi: 10.1074/jbc.m300470200

Figure Lengend Snippet: FIG. 3. Expression and cellular localization of CXCR3 in the caprine conceptuses. A, levels of CXCR3 mRNA in the pregnant endometrium (D14, D17, and D20) and day 17 conceptuses (Con) were examined using RT-PCR. B, presence of CXCR3 mRNA in the caprine conceptus was confirmed by Northern blot analysis. For A and B, duplicate samples were analyzed for each day, and three independent experiments were performed for each day. C, immunofluorescence mi- croscopy was performed on day 17 conceptuses using anti-human CXCR3 monoclonal antibody (a) or normal mouse IgG as negative control (d), and nuclei stained with propidium iodide were shown in the same field (b and e). c, combined fluorescence image resulting from anti-CXCR3 and propidium iodide was shown. Scale bar, 100 m. Three independent experiments were performed for each treatment.

Article Snippet: Nonspecific binding was blocked by a treatment with Block Ace at room temperature for 1 h, and slides were then incubated with a mouse monoclonal antibody to human CXCR3 (10 g/ml, R&D Systems Inc., Minneapolis, MN) or normal mouse IgG (Sigma) at 4 °C for 12 h. After the primary antibody incubation, slides were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (15 g/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. Nuclei were stained with propidium iodide (2 g/ml, Sigma).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Northern Blot, Immunofluorescence, Negative Control, Staining, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation

doi: 10.1074/jbc.m300470200

Figure Lengend Snippet: FIG. 4. Binding of rcIP-10 to the ca- prine trophoblast cells. A, recombinant proteins, cIP-10, GST, and caprine lym- photactin (Lymphotactin-GST), were bi- otinylated using the EZ-link biotinylation reagent and the labeled proteins (2 g/ lane) were detected with streptavidin- horseradish peroxidases. B, expressions of IP-10 receptor CXCR3 mRNA and lym- photactin receptor XCR1 mRNA in the caprine conceptus, endometrium or PBMC were confirmed by RT-PCR. Con, conceptus on day 17 of pregnancy. Endo, endometrium on day 17 of pregnancy. PB- MCs, peripheral blood mononuclear cells. C, biotinylated proteins were incubated with day 17 caprine conceptuses and vi- sualized with streptavidin-horseradish peroxidases. Scale bar, 200 m. Duplicate samples were tested for each treatment, and three independent experiments were performed.

Article Snippet: Nonspecific binding was blocked by a treatment with Block Ace at room temperature for 1 h, and slides were then incubated with a mouse monoclonal antibody to human CXCR3 (10 g/ml, R&D Systems Inc., Minneapolis, MN) or normal mouse IgG (Sigma) at 4 °C for 12 h. After the primary antibody incubation, slides were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (15 g/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. Nuclei were stained with propidium iodide (2 g/ml, Sigma).

Techniques: Binding Assay, Recombinant, Labeling, Reverse Transcription Polymerase Chain Reaction, Incubation

Journal: Journal of Biological Chemistry

Article Title: Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation

doi: 10.1074/jbc.m300470200

Figure Lengend Snippet: FIG. 5. Stimulation of rcIP-10 on the migration of CXCR3-expressing conceptus cells. A, left, Northern blot analysis of CXCR3 mRNA. RNA was extracted from HTS-1 cells () or those that had been transiently transfected with parental pcDNA3.1 (Mock) or pcDNA3.1-caprine CXCR3 (CXCR3). Right, biological activity of rcIP-10 to HTS-1 cells that had been transfected (circle) or not transfected (square) with CXCR3 expression plasmid was tested using chemotaxis assay. B, effect of rcIP-10 on migratory activity of HTS-1 cells that had been transfected (black bar) or not transfected (white bar) with the CXCR3 construct was examined. These cells were treated () or not treated () with rcIP-10 (20 ng/ml), which had been pretreated with the anti-caprine IP-10 antibody (IP-10, 30 g/ml) or control rabbit IgG (IgG, 30 g/ml). Ratio of migrated cells was calculated as the number of migrated cells treated with rcIP-10 divided by the number of migrated cells without the rcIP-10 treatment. An asterisk indicates a significant difference (p 0.05). C, effect of rcIP-10 on migratory activity of primary caprine trophoblast cells on day 17 of pregnancy was examined. For the blocking experiments, rcIP-10 (20 ng/ml) was preincubated with 30 g/ml of anti-IP-10 antibody (IP-10) or normal rabbit IgG (IgG), which was then subjected to chemotaxis assay. Bars represent LSM S.E. An asterisk indicates a significant difference (p 0.05). Three replicates were performed for each treatment, and three independent experiments were performed for each treatment.

Article Snippet: Nonspecific binding was blocked by a treatment with Block Ace at room temperature for 1 h, and slides were then incubated with a mouse monoclonal antibody to human CXCR3 (10 g/ml, R&D Systems Inc., Minneapolis, MN) or normal mouse IgG (Sigma) at 4 °C for 12 h. After the primary antibody incubation, slides were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (15 g/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. Nuclei were stained with propidium iodide (2 g/ml, Sigma).

Techniques: Migration, Expressing, Northern Blot, Transfection, Activity Assay, Plasmid Preparation, Chemotaxis Assay, Construct, Control, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation

doi: 10.1074/jbc.m300470200

Figure Lengend Snippet: FIG. 6. Promotion of rcIP-10 on the adhesion of caprine trophoblast cells to fibronectin or endometrial epithelial cells. A, adhesions of primary caprine trophoblast cells (day 17) stimulated () or not stimulated () with rcIP-10 (20 ng/ml) to the chamber coated with collagen I, fibronectin or non-substrate (non-coated) were assessed by using adhesion assay. B, effect of rcIP-10 (20 ng/ml) on the attachment of primary caprine trophoblast cells (day 17) to fibronectin was also assessed using the adhesion assay. Blocking experiment was performed using the pretreatment of rcIP-10 (20 ng/ml) with anti-caprine IP-10 antibody (IP-10, 30 g/ml) or normal rabbit IgG (30 g/ml), which was then added to the cells. The remaining treatments, 50 mM Arg-Gly-Asp (RGD) peptide, 50 mM Arg-Gly-Glu (RGE) peptide, or 5 mM EDTA, were applied to the trophoblast cells, which were then subjected to chemotaxis assay. C, HTS-1 transiently transfected with pcDNA3.1-caprine CXCR3 (solid column) or parental pcDNA3.1 (open column) were subjected to the fibronectin adhesion assay. Treatments with 20 ng/ml rcIP-10, 30 g/ml of anti-IP-10 antibody (IP-10) or normal rabbit IgG (IgG), 50 mM RGD, or 50 mM RGE peptides, or 5 mM EDTA were applied as aforementioned. D, HTS-1 cells transiently transfected with pcDNA3.1-caprine CXCR3 (solid column) or parental pcDNA3.1 (open column) were subjected to the adhesion assay with endometrial epithelial cells. Bars represent LSM S.E. and an asterisk(s) indicates a significant difference (p 0.05). Triplicate samples were examined for each treatment, and three independent experiments were performed.

Article Snippet: Nonspecific binding was blocked by a treatment with Block Ace at room temperature for 1 h, and slides were then incubated with a mouse monoclonal antibody to human CXCR3 (10 g/ml, R&D Systems Inc., Minneapolis, MN) or normal mouse IgG (Sigma) at 4 °C for 12 h. After the primary antibody incubation, slides were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (15 g/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. Nuclei were stained with propidium iodide (2 g/ml, Sigma).

Techniques: Cell Adhesion Assay, Blocking Assay, Chemotaxis Assay, Transfection

Journal: Journal of Biological Chemistry

Article Title: Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon γ-inducible Protein 10 kDa (IP-10), during Early Gestation

doi: 10.1074/jbc.m300470200

Figure Lengend Snippet: FIG. 7. Expression of integrin sub- units in the caprine trophoblast cells stimulated by rcIP-10. Upper, RT-PCR analysis of expressions of integrin 5, V, 1, 3, and 5 subunits, and G3PDH mRNAs. Recombinant cIP-10 (20 ng/ml) pretreated (IP-10 Abs) or not pre- treated (IP-10) with anti-IP-10 antibody (30 g/ml) was applied to HTS-1 cells that had been transiently transfected with pcDNA3.1-caprine CXCR3. Lower, densi- tometric analysis of PCR products. Bars represent LSM S.E., and differences in integrin mRNA levels among treatments were shown with a superscript.

Article Snippet: Nonspecific binding was blocked by a treatment with Block Ace at room temperature for 1 h, and slides were then incubated with a mouse monoclonal antibody to human CXCR3 (10 g/ml, R&D Systems Inc., Minneapolis, MN) or normal mouse IgG (Sigma) at 4 °C for 12 h. After the primary antibody incubation, slides were treated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (15 g/ml, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at room temperature for 1 h. Nuclei were stained with propidium iodide (2 g/ml, Sigma).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Recombinant, Transfection

Journal: Clinical Cancer Research

Article Title: Role of CXCR3 Ligands in IL-7/IL-7Rα-Fc–Mediated Antitumor Activity in Lung Cancer

doi: 10.1158/1078-0432.ccr-10-3346

Figure Lengend Snippet: Figure 4. CXCR3 pathway is required for IL-7/IL-7Ra-Fc–mediated antitumor activity. For in vivo neutralizations, mice bearing 5-day established subcutaneous tumors were treated with IL-7/IL7Ra-Fc on days 5 and 10. Twenty four hours prior to IL-7/IL7Ra-Fc treatment, and then 3 times per week, mice were injected i.p. individually with the respective specific or appropriate control antibodies for the duration of the experiment. A and B, neutralization of CXCL9 (A) or CXCL10 (B) or IFNg (B) reversed the antitumor benefit of IL-7/IL-7Ra-Fc [bottom, photograph of tumors from diluents, IL-7/IL-7Ra-Fc, and IL-7/IL-7Ra-Fc þ anti-CXCL9, or anti-CXCL10 or anti-IFNg (IL-7/IL-7Ra-Fc þ control Ab was the same as IL-7/IL-7Ra-Fc treated group, data not shown)]. C, CXCL9, CXCL10, and IFNg were reduced in tumors (day 15) following treatment of the tumor bearing mice with IL-7/IL-7Ra-Fc and the respective neutralizing antibodies: anti-CXCL9, or anti-CXCL10 or anti-IFNg antibody in comparison to control Abs. D, neutralization of CXCL9, CXCL10, or IFNg reduced the frequency of CXCR3 activated CD8 T cells in the tumor (day 15). Data; mean SEM; *, P < 0.01 for IL-7/IL-7Ra-Fc þ control Ab compared with IL-7/IL- 7Ra-Fc þ anti-cytokine Ab or Diluent groups (n ¼ 6/group).

Article Snippet: Anti-mouse mAbs to CXCR3 (220803), IL-7Ra-Fc chimeric molecule (747-MR; endotoxin level: less than 1.0 EU/mg of protein by LAL method), ELISA antibody pairs for murine IFNg , CXCL9, CXCL10, and IL12 were purchased from R&D Systems, sensitivity of 3 to 5 pg/mL.

Techniques: Activity Assay, In Vivo, Injection, Control, Neutralization, Comparison

Journal: The Korean journal of internal medicine

Article Title: Therapeutic effect of multiple functional minicircle vector encoding anti-CD25/IL-10/CXCR3 in allograft rejection model.

doi: 10.3904/kjim.2021.299

Figure Lengend Snippet: Figure 1. Diagram of the novel multiple target-directed agent (MTA), which was constructed as an anti-CD25 antibody conju- gated to interleukin-10 (IL-10) and C-X-C motif chemokine recep- tor 3 (CXCR3) in turn at the end of the Fab region. MTA structure designed by the fusion of the anti-CD25 antibody and the dimer form of IL-10 via short flexible linkers, called (G4S1)3 linker. CXCR3 was connected with IL-10 using the (G4S1)2 linker. (G4S1)3, (GG- GGS)3; (G4S1)2, (GGGGS)2; mIL-10, mouse intrleukin-10; mCXCR3, mouse C-X-C motif chemokine receptor 3; ECD1, extracellular domain 1.

Article Snippet: Briefly, a 96-well microtiter plate was coated with each commercial anti-mouse IL-10 (MAB417-100, R&D Systems, Minneapolis, MN, USA; at a concentration of 1 mg/mL) or anti-mouse CXCR3 (NBP2-41250, Novus Biologicals, Centennial, CO, USA; at a concentration of 1 mg/mL) in coating buffer and was incubated overnight at 4°C.

Techniques: Construct

Journal: The Korean journal of internal medicine

Article Title: Therapeutic effect of multiple functional minicircle vector encoding anti-CD25/IL-10/CXCR3 in allograft rejection model.

doi: 10.3904/kjim.2021.299

Figure Lengend Snippet: Figure 3. Detection of multiple target-directed agent (MTA) derived from minicircles (MCs) in vitro. (A) Fluorescence images of the ex- pression of red fluorescence protein (RFP). HEK293T cells were transfected with the indicated DNA, and RFP expression was analyzed using fluorescence microscopy at 3 days post-transfection. HEK293T cells that were not transfected with DNA were used as a nil group. Scale bar = 50 μm. (B, C, D) Concentrations of anti-CD25, interleukin-10 (IL-10), and C-X-C motif chemokine receptor 3 (CXCR3) in con- ditioned media of cells transfected with MC-mock or MC-MTA. Protein secretion from HEK293T cells was assessed using enzyme-linked immunosorbent assay (ELISA) at 3 days after transfection. Values are mean ± standard error. All results are representative of at least three independent experiments. DIC, differential interference contrast; PP, parent plasmid. ap < 0.05 vs. MC-mock.

Article Snippet: Briefly, a 96-well microtiter plate was coated with each commercial anti-mouse IL-10 (MAB417-100, R&D Systems, Minneapolis, MN, USA; at a concentration of 1 mg/mL) or anti-mouse CXCR3 (NBP2-41250, Novus Biologicals, Centennial, CO, USA; at a concentration of 1 mg/mL) in coating buffer and was incubated overnight at 4°C.

Techniques: Derivative Assay, In Vitro, Fluorescence, Transfection, Expressing, Microscopy, Enzyme-linked Immunosorbent Assay, Plasmid Preparation