Journal: BMC Immunology

Article Title: New perspectives for natural antimicrobial peptides: application as antinflammatory drugs in a murine model

doi: 10.1186/1471-2172-13-61

Figure Lengend Snippet: (A) TNF-α, IFN-γ, IL-10 mRNA expression levels in J774 cells stimulated with LPS or LTA (0,1,1 or 10 μg/ml) for 3 hours. ( B ) J774 cells treated with gentamicin (5 μg/ml) or MIX (RJI-C 9 μg/ml + TB-KK 6 μg/ml) or ASA (5 μg/ml) for 3 hours. ( C ) J774 cells stimulated with LPS (10 μg/ml) for 3 hours and treated with gentamicin (5 μg/ml) or MIX (RJI-C 9 μg/ml + TB-KK 6 μg/ml) or ASA (5 μg/ml) for further 3 hours. ( D ) Western blot analysis of COX-2 in J774 cell line. Lane 1 – 3 : J774 cells + LPS(10 μg/ml); Lane 4 – 6 : J774 cells + LPS (10 μg/ml) + inactive peptide (RJII-C 15 μg/ml); Lane 7 – 9 : J774 cells + LPS (10 μg/ml) + ASA (5 μg/ml); Lane 10 – 12 : J774 cells + LPS (10 μg/ml) + MIX (RJI-C 9 μg/ml + TB-KK 6 μg/ml; Lane 13 – 14 : J774 cells + LPS (10 μg/ml) + gentamicin (5 μg/ml). ( E ) TNF-α, IFN-γ, IL-10 mRNA expression levels in kidney of mice (3mice/group) stimulated with LPS (250 μg, ~10 mg/Kg) for 3 hours; stimulated with LPS (250 μg, ~10 mg/Kg) for 3 hours and treated with gentamicin (5 μg/mouse) or MIX (RJI-C 9 μg/mouse + TB-KK 6 μg/mouse) or ASA (5 μg/mouse) for 3 hours. Values were normalized with GAPDH and compared to untreated control. *P <0.05, **p < 0.01; ***p < 0.001, Student’s t test gentamicin vs MIX and gentamicin vs ASA.

Article Snippet: The supernatants from these cells (100 μl/well) were transferred into the wells of a plate previously coated with mouse anti-human TNF-α (BD Pharmingen; 50 μl diluted 2 x 10-3/well) or mouse anti-human IFN-γ (Biosciences, 50 μl diluted 2 x 10-3/well) along with a second dose of anti IFN- γ or TNF- α, HRP-labelled rabbit anti mouse IgG diluted 10 -3 (100 μl/well) and TMB peroxidase substrate (BIORAD; 100 μL/well), in the order.

Techniques: Expressing, Western Blot

Journal: BMC Immunology

Article Title: New perspectives for natural antimicrobial peptides: application as antinflammatory drugs in a murine model

doi: 10.1186/1471-2172-13-61

Figure Lengend Snippet: (A-C) TNF-α, IFN-γ, IL-10 mRNA expression levels in infected mice with Staphylococcus epidermidis (108 CFU/mouse) or infected with Staphylococcus epidermidis (108 CFU/mouse) and treated with the MIX (RJI-C at 9 μg/mouse and TB-KK at 6 μg/mouse) or gentamicin (5 μg/mouse) at 3(A), 6 (B) and 9 (C) hours after treatment. Values were normalized with GAPDH and compared to untreated control. *P <0.05, **p < 0.01; ***p < 0.001, Student’s t test gentamicin vs MIX.

Article Snippet: The supernatants from these cells (100 μl/well) were transferred into the wells of a plate previously coated with mouse anti-human TNF-α (BD Pharmingen; 50 μl diluted 2 x 10-3/well) or mouse anti-human IFN-γ (Biosciences, 50 μl diluted 2 x 10-3/well) along with a second dose of anti IFN- γ or TNF- α, HRP-labelled rabbit anti mouse IgG diluted 10 -3 (100 μl/well) and TMB peroxidase substrate (BIORAD; 100 μL/well), in the order.

Techniques: Expressing, Infection

Journal: BMC Immunology

Article Title: New perspectives for natural antimicrobial peptides: application as antinflammatory drugs in a murine model

doi: 10.1186/1471-2172-13-61

Figure Lengend Snippet: (A-B) Bacterial load in spleen and kidneys of animals (24/groups) infected with Staphylococcus epidermidis (107 CFU/mouse; rumble line); infected with Staphylococcus epidermidis(107 CFU/mouse) and treated with the MIX (RJI-C at 9 μg/mouse and TB-KK at 6 μg/mouse; square line) or gentamicin (5 μg/mouse; triangle line) *P <0.05, **p < 0.01; ***p < 0.001, Student’s t test gentamicin vs MIX. ( C ) TNF-α, IFN-γ, IL-10 mRNA expression levels were quantified, at the indicated time points, in mice infected with Staphylococcus epidermidis (10 7 CFU/mouse) or infected with Staphylococcus epidermidis (10 7 CFU/mouse) and treated with three different doses (I,II,III) of the MIX (RJI-C at 9 μg in 100 μl/mouse and TB-KK at 6 μg in 100 μl/mouse) or gentamicin (5 μg in 100 μl/mouse). Values were normalized with GAPDH and compared to untreated control. *P <0.05, **p < 0.01; ***p < 0.001, Student’s t test gentamicin vs MIX.

Article Snippet: The supernatants from these cells (100 μl/well) were transferred into the wells of a plate previously coated with mouse anti-human TNF-α (BD Pharmingen; 50 μl diluted 2 x 10-3/well) or mouse anti-human IFN-γ (Biosciences, 50 μl diluted 2 x 10-3/well) along with a second dose of anti IFN- γ or TNF- α, HRP-labelled rabbit anti mouse IgG diluted 10 -3 (100 μl/well) and TMB peroxidase substrate (BIORAD; 100 μL/well), in the order.

Techniques: Infection, Expressing

Journal: STAR Protocols

Article Title: Protocol for measuring killing capacity and intracellular cytokine production of human HIV antigen-specific CD8 T cells using flow cytometry

doi: 10.1016/j.xpro.2024.103231

Figure Lengend Snippet:

Article Snippet: BD Pharmingen Alexa Fluor 700 mouse anti-human TNF, dilution: 1:160 , BD Biosciences , 561023.

Techniques: Blocking Assay, Recombinant, Staining, Flow Cytometry, Infection, Cell Isolation, Software

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus enhances TNF-α and IL-12 production of human MDDCs. Human immature MDDCs were infected with recombinant ALVAC virus expressing human CD40L called vA3131-2, an ALVAC-HIV vaccine called vCP205, or a parental control, ALVAC II at an MOI of 10, or incubated with medium alone, or with CD40L trimer (2 μg/ml) as a positive control at 37°C for 24 h. The production of TNF-α and IL-12 was determined by intracellular staining flow cytometry. (A) Data are taken from the HIV-1-infected participant #1 and are representative of experiments with MDDCs derived from two HIV-1-infected and two HIV-1-uninfected individuals. The numbers in each gate represent the percentage of TNF-α or IL-12-positive MDDCs in total MDDCs. (B) Pooled data from all participants are shown. Data shown are mean±SEM. **: P<0.01.

Article Snippet: Then the cells were permeablized with Cytofix/Cytoperm solution (BD Biosciences) and stained with APC-mouse anti-human TNF-α mAb and PE-mouse anti-human IL-12 mAb (BD Biosciences).

Techniques: Recombinant, Infection, Expressing, Incubation, Positive Control, Staining, Flow Cytometry, Derivative Assay

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Structure and restriction sites of TM-TNF-α. TM-TNF-α including the extracellular, transmembrane and cytoplasmic domains. Hydrolysis with TACE, results in a mature TNF-α consisting of 157 amino acid residues, known as s-TNF-α. TM, transmembrane region; TNF, tumor necrosis factor; TACE, TNF-α converting enzyme.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques:

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Purified reverse transcription-polymerase chain reaction products analyzed by 1% agarose gel electrophoresis. M, wide-range DNA marker (100–6,000 bp); lanes 1 and 2, transmembrane-tumor necrosis factor-α cDNA (obtained from HL-60 cells and purified).

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Purification, Reverse Transcription, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Construction and identification of pcDNA3.1-TM-enterokinase-TNF-α and pcDNA3.1-TM-FactorXa-TNF-α vectors. (A) Overlapping PCR fragments analyzed by 2% agarose gel electrophoresis. M, wide-range DNA marker (100–6,000 bp); lane 1, the transmembrane domain fragment with an enterokinase restriction site; lanes 2 and 4, soluble-TNF-α fragment; lane 3, the transmembrane domain fragment containing a FactorXa restriction site. (B) Overlapping PCR products analyzed by 2% agarose gel electrophoresis. M, wide range DNA marker (100–6,000 bp); lanes 1 and 2, fragment with an enterokinase restriction site; lanes 3 and 4, fragment with a FactorXa restriction site. (C) Restriction enzyme ( Eco RI and Nhe I) digestion of positive cloning vector analyzed by 2% agarose gel electrophoresis. M, wide-range DNA marker (100–6,000 bp); lanes 1 and 2, the fragment with an enterokinase restriction site; lanes 3 and 4, the fragment with a FactorXa restriction site. (D) DNA sequencing analysis of the positively cloned recombinant pcDNA3.1-TM-enterokinase-TNF-α plasmid. (E) DNA sequencing analysis of the positively cloned pcDNA3.1-TM-FactorXa-TNF-α recombinant plasmid. TM, transmembrane; TNF-α, tumor necrosis factor-α; PCR, polymerase chain reaction.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Agarose Gel Electrophoresis, Marker, Cloning, Plasmid Preparation, DNA Sequencing, Clone Assay, Recombinant, Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Reverse transcription-polymerase chain reaction products analyzed by 2% agarose gel electrophoresis. M, wide-range DNA marker (100–6,000 bp); lane 1, transfection with pcDNA3.1-TM-enterokinase-TNF-α plasmid; lane 2, transfection with pcDNA3.1-TM-Factor Xa-TNF-α plasmid; lane 3, control (non-transfected 3T3 cells); TM, transmembrane; TNF-α, tumor necrosis factor-α.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Transfection, Plasmid Preparation, Control

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Expression of the TNF-α proteins as detected by western blotting. Lane 1, transfection of 3T3 cells with pcDNA3.1-TM-enterokinase-TNF-α plasmid; lane 2, transfection of 3T3 cells with pcDNA3.1-TM-FactorXa-TNF-α plasmid; lane 3, control. TM, transmembrane; TNF-α, tumor necrosis factor-α.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Detection of TNF-α in extracellular digestive enzymatic solutions from 3T3 cells that were transfected with pcDNA3.1-TM-enterokinase-TNF-α and pcDNA3.1-TM-FactorXa-TNF-α by SDS-PAGE and western blotting. (A) Digestion solution analyzed by SDS-PAGE. M, protein marker; lane 1, transfection with pcDNA3.1-TM-FactorXa-TNF-α plasmid; lane 2, transfection with pcDNA3.1-TM-enterokinase-TNF-α plasmid; lane 3, cell medium digested by FactorXa; lane 4, cell medium digested by enterokinase; lane 5, control. (B) Detection of TNF-α following digestion by western blotting. Lane 1, transfection with pcDNA3.1-TM-enterokinase-TNF-α plasmid; lane 2, transfection with pcDNA3.1-TM-FactorXa-TNF-α plasmid; lane 3, control. TM, transmembrane; TNF-α, tumor necrosis factor-α.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Transfection, SDS Page, Western Blot, Marker, Plasmid Preparation, Control

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Cytotoxicity of hTNF-α and extracellular fluid TNF-α treatment of L929 cells. **P<0.01 vs. 0 pmol/l hTNF-α. hTNF-α, human tumor necrosis factor-α. TM, transmembrane; TNF-α, tumor necrosis factor-α.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques:

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Comparison of the specific activity of different proteins.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Comparison, Activity Assay

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: MTT assay results demonstrating the effect of TNF-α on L929 cell viability.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: MTT Assay, Standard Deviation, Inhibition, Negative Control

Journal: Molecular Medicine Reports

Article Title: Construction and characterization of a transmembrane eukaryotic expression vector based on the membrane domain structure of TNF-α

doi: 10.3892/mmr.2017.6692

Figure Lengend Snippet: Morphological alterations of L929 cells following treatment with TNF-α for 24 h. (A) Physiological saline negative control. (B) hTNF-α-treated positive control. (625 pmol/l) (C) Extracellular fluid TNF-α (625 pmol/l). hTNF-α, human tumor necrosis factor-α. Magnification, ×100. hTNF-α, human tumor necrosis factor-α.

Article Snippet: The membranes were blocked with 5% non-fat milk in phosphate-buffered saline containing Tween-20 (PBST) for 2 h at room temperature, and were subsequently incubated with mouse anti-human TNF-α antibody (cat. no. 551220; dilution, 1:1,000 in Primary Antibody Dilution Buffer; Beyotime Institute of Biotechnology) overnight at 4°C.

Techniques: Saline, Negative Control, Positive Control