Journal: Journal of Virology

Article Title: Poxvirus Tumor Necrosis Factor Receptor (TNFR)-Like T2 Proteins Contain a Conserved Preligand Assembly Domain That Inhibits Cellular TNFR1-Induced Cell Death

doi: 10.1128/jvi.02449-05

Figure Lengend Snippet: FIG. 2. Poxvirus T2-like molecules contain a conserved PLAD. Sequence similarity analysis of the N-terminal region of the myxoma virus (MV), Shope fibroma virus (SFV), VV strain Lister and Copenhagen, camelpox virus (CMLV), variola major virus strain Bangladesh (VAR bsh major), variola minor virus strain Garcia (VAR gar minor), monkeypox virus (MPV), and ectromelia virus (EV) T2 ORFs and human TNFR1 and TNFR2 reveals a highly conserved PLAD that overlaps the first CRD. Identical amino acids (bold) and conserved amino acid differences (gray) are indicated.

Article Snippet: Alternatively, lysates were analyzed directly by immunoblotting with B5 anti-M-T2, H5 anti-TNFR1, or C20 anti-TNFR2 antibody (Santa Cruz).

Techniques: Sequencing, Virus

Journal: Journal of Virology

Article Title: Poxvirus Tumor Necrosis Factor Receptor (TNFR)-Like T2 Proteins Contain a Conserved Preligand Assembly Domain That Inhibits Cellular TNFR1-Induced Cell Death

doi: 10.1128/jvi.02449-05

Figure Lengend Snippet: FIG. 4. Immunoprecipitation analysis of M-T2 and TNFR. HEK 293T cells were cotransfected with pcDNA3-TNFR1 or pcDNA3- TNFR2 and either pcDNA3-M-T2myc, pcDNA3-T2PLADmyc, or pcDNA3-LacZ. TNFR1 was immunoprecipitated with anti-TNFR1- specific H5 antibody, TNFR2 was immunoprecipitated with TNFR2- specific C20 antibody, and TNFR-associated M-T2 was detected with anti-M-T2 B5 antibody. IP, immunoprecipitation; WB, Western im- munoblotting.

Article Snippet: Alternatively, lysates were analyzed directly by immunoblotting with B5 anti-M-T2, H5 anti-TNFR1, or C20 anti-TNFR2 antibody (Santa Cruz).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Virology

Article Title: Poxvirus Tumor Necrosis Factor Receptor (TNFR)-Like T2 Proteins Contain a Conserved Preligand Assembly Domain That Inhibits Cellular TNFR1-Induced Cell Death

doi: 10.1128/jvi.02449-05

Figure Lengend Snippet: FIG. 6. Cell-associated M-T2 and TNF--binding. (A) Flow cytom- etry analysis of human TNF- binding to the surface of HEK 293T cells transfected with TNFR1, TNFR1 plus M-T2myc, or TNFR1 plus M-T2PLADmyc (top row) or with TNFR2, TNFR2 plus M-T2myc, or TNFR2 plus M-T2PLADmyc (second row) at 24 h posttransfec- tion. Shown is the surface fluorescence of bound TNF-, detected with anti-human TNF-–PE antibody, after 10 min of culture with (red, unfilled histograms) or without (black, unfilled histograms) recombi- nant human TNF-. Isotype control staining (filled histograms) and surface expression levels of TNFR1 and TNFR2 24 h after transfection are also shown, as are overlays of TNF- staining in each transfec- tion (orange, TNFR; green, TNFR plus M-T2; blue, TNFR plus M-T2PLAD). Data shown are representative of independently re- peated experiments. (B) Surface detection of TNFR1-CFP and M-T2myc protein at 24 h posttransfection on unpermeabilized U20S cells (TNFR1-CFP, M-T2myc [Cy3], and merged images of both). (C) Cell-associated M-T2myc and M-T2PLADmyc binding to rabbit TNF-. M-T2myc- and M-T2PLADmyc-containing HEK 293T trans- fected-cell lysates were mixed with Vero cell lysates containing rabbit TNF- expressed by recombinant VV. M-T2myc-bound rabbit TNF- was detected by immunoprecipitation (IP) with anti-myc antibody and Western immunoblotting (WB) with rabbit TNF--specific antibody (as indicated). Expression of M-T2, M-T2PLAD, and rabbit TNF- was confirmed by Western immunoblotting.

Article Snippet: Alternatively, lysates were analyzed directly by immunoblotting with B5 anti-M-T2, H5 anti-TNFR1, or C20 anti-TNFR2 antibody (Santa Cruz).

Techniques: Binding Assay, Transfection, Control, Staining, Expressing, Recombinant, Immunoprecipitation, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Decreased progranulin levels in patients and rats with subarachnoid hemorrhage: a potential role in inhibiting inflammation by suppressing neutrophil recruitment

doi: 10.1186/s12974-015-0415-4

Figure Lengend Snippet: ELISA analysis of PGRN, MPO, IL-β, and TNF-α in the CSF of patients. The concentration of PGRN decreased after SAH and was lowest in the 1–3 days group ( a ). However, the concentration of MPO, IL-β, and TNF-α increased after SAH and was highest in the 1–3 days group ( b – d ). Data are expressed as the mean ± SEM from six rats. * p < 0.05 compared with the control group, ns p > 0.05 compared with the control group

Article Snippet: The inflammatory cytokine levels of brain tissue were quantified using enzyme-linked immunosorbent assay (ELISA) kits specific for rats according to the manufacturer’s instructions (PGRN, P28799, from Raybiotech, USA; MPO, ab119605, from Abcam, USA; tumor necrosis factor-α (TNF-α), 950.090.096 and 865.000.096, from Diaclone Research, France; IL-1β, 850.006.096 and 670.040.096, from Diaclone Research, France).

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

Journal: Journal of Neuroinflammation

Article Title: Decreased progranulin levels in patients and rats with subarachnoid hemorrhage: a potential role in inhibiting inflammation by suppressing neutrophil recruitment

doi: 10.1186/s12974-015-0415-4

Figure Lengend Snippet: ELISA analysis of IL-β and TNF-α in the brain cortex of rats 24 h after SAH. The concentrations of IL-β ( a ) and TNF-α ( b ) significantly increased at 24 h after SAH and decreased after administration of r-PGRN. No difference was detected between the untreated SAH group and the PBS-treated SAH group. Data are expressed as the mean ± SEM from six rats. *** p < 0.001 compared with the sham group, ns p > 0.05 compared with the SAH group, ## p < 0.01 compared with the SAH group

Article Snippet: The inflammatory cytokine levels of brain tissue were quantified using enzyme-linked immunosorbent assay (ELISA) kits specific for rats according to the manufacturer’s instructions (PGRN, P28799, from Raybiotech, USA; MPO, ab119605, from Abcam, USA; tumor necrosis factor-α (TNF-α), 950.090.096 and 865.000.096, from Diaclone Research, France; IL-1β, 850.006.096 and 670.040.096, from Diaclone Research, France).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Life sciences

Article Title: Aging-related hyperphosphatemia triggers the release of TNF-α from macrophages, promoting indicators of sarcopenia through the reduction of IL-15 expression in skeletal muscle.

doi: 10.1016/j.lfs.2025.123507

Figure Lengend Snippet: Fig. 3. Conditioned medium collected from BGP-treated macrophages impairs myogenic differentiation of myoblasts. A,B,C) C2C12 cells were incubated with 2 % HS to promote myogenic differentiation for 72 h in the presence or absence (Control, CT) of 5 % conditioned medium collected from macrophages treated with β-glycerophosphate (BGP) (CM(BGP)) for 6, 8 or 24 h or the vehicle (CM). MHC (A), MyoG (B) and fibronectin (FN) (C) expressions were analysed by western blotting. Representative blots are shown in the left panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 12 experiments. D,E) C2C12 were incubated in the same conditions as in panels A, B and C in the presence or absence of a TNF-α neutralising antibody (Ab TNF, 2 μg/mL) or IgG antibody as a control (Ab IgG). MHC (D), and FN (E) expressions were analysed by western blotting. Representative blots are shown in the upper panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 8 experiments. *p < 0.05 vs CT. #p < 0.05 vs CM. $p < 0.05 vs CM(BGP).

Article Snippet: To analyse the role of the cytokine TNF-α in the conditioned medium, certain experiments were performed in the presence of a TNF-α neutralising antibody (2 μg/ mL) (MAB4101, R&D Systems, Minneapolis, MN, USA) or an IgG antibody (sc-2025, Santa Cruz BioTechnology, Sta.

Techniques: Incubation, Control, Western Blot

Journal: Life sciences

Article Title: Aging-related hyperphosphatemia triggers the release of TNF-α from macrophages, promoting indicators of sarcopenia through the reduction of IL-15 expression in skeletal muscle.

doi: 10.1016/j.lfs.2025.123507

Figure Lengend Snippet: Fig. 4. Conditioned medium collected from BGP-treated macrophages induces senescence in myoblasts. A,B) C2C12 cells were incubated in the presence or absence (Control, CT) of 3 % conditioned medium collected from macrophages treated with β-glycerophosphate (BGP) (CM(BGP)) for 6, 8 or 24 h or the vehicle (CM) for 72 h. C,D) C2C12 cells were incubated in the in the same conditions as in panels A and B in the presence or absence of a TNF-α neutralising antibody (Ab TNF, 2 μg/mL) or IgG antibody as a control (Ab IgG). A,C) Senescence-associated β-galactosidase activity was analysed by confocal microscopy using the fluorogenic substrate C12FDG. Representative images are shown in the left panels. Bar graphs represent the densitometric analysis of the green fluorescence intensity and results are expressed as the mean ± SEM from 7 (A) or 5 (B) experiments. B,D) p21 expression was analysed by western blotting. Representative blots are shown in the left panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 5 experiments. *p < 0.05 vs CT. #p < 0.05 vs CM. $p < 0.05 vs CM(BGP). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To analyse the role of the cytokine TNF-α in the conditioned medium, certain experiments were performed in the presence of a TNF-α neutralising antibody (2 μg/ mL) (MAB4101, R&D Systems, Minneapolis, MN, USA) or an IgG antibody (sc-2025, Santa Cruz BioTechnology, Sta.

Techniques: Incubation, Control, Activity Assay, Confocal Microscopy, Fluorescence, Expressing, Western Blot

Journal: Life sciences

Article Title: Aging-related hyperphosphatemia triggers the release of TNF-α from macrophages, promoting indicators of sarcopenia through the reduction of IL-15 expression in skeletal muscle.

doi: 10.1016/j.lfs.2025.123507

Figure Lengend Snippet: Fig. 6. IL-15 prevents the signs of sarcopenia in myoblasts promoted by the conditioned medium collected from BGP-treated macrophages. A) C2C12 cells were treated with 10 mM BGP for 6, 8 or 24 h. B) C2C12 cells were incubated in the presence or absence (Control, CT) of 5 % conditioned medium collected from macrophages treated with β-glycerophosphate (BGP) (24 h) (CM(BGP)) or the vehicle (CM) for 24 h. C) C2C12 cells were incubated in the same conditions as in panel B in the presence or absence of a TNF-α neutralising antibody (Ab TNF-α, 2 μg/mL) or IgG antibody as a control (Ab IgG). D) C2C12 cells were treated with different doses of TNF-α for 24 h. A,B,C,D) IL-15 expression was measured by RT-qPCR using GAPDH as endogenous control. Bar graphs represent the mean ± SEM from 5 (A), 5 (B), 5 (C) or 6 (D) experiments. E,F) C2C12 cells were cultured with 2 % HS to promote myogenic differentiation and incubated with 5 % CM or CM(BGP) for 72 h in the presence or absence of different doses of recombinant IL-15 (100–1000 ng/mL). MHC (E) and fibronectin (FN) (F) expressions were analysed by western blotting. Representative blots are shown in the left panels. Bar graphs represent the densitometric analysis of the bands and results are expressed as the mean ± SEM from 5 experiments. G) C2C12 cells were incubated with 3 % CM or CM(BGP) for 72 h in the presence or absence of different doses of recombinant IL-15. Senescence- associated β-galactosidase activity was analysed by confocal microscopy using the fluorogenic substrate C12FDG. Representative images are shown in the left panels. Bar graphs represent the densitometric analysis of the green fluorescence intensity and results are expressed as the mean ± SEM from 4 experiments. *p < 0.05 vs CT. #p < 0.05 vs CM. $p < 0.05 vs CM(BGP). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To analyse the role of the cytokine TNF-α in the conditioned medium, certain experiments were performed in the presence of a TNF-α neutralising antibody (2 μg/ mL) (MAB4101, R&D Systems, Minneapolis, MN, USA) or an IgG antibody (sc-2025, Santa Cruz BioTechnology, Sta.

Techniques: Incubation, Control, Expressing, Quantitative RT-PCR, Cell Culture, Recombinant, Western Blot, Activity Assay, Confocal Microscopy, Fluorescence

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Measuring T cell functions by flow cytometry vs. ELISPOT. Left panel: For the detection of secretory products by ICS, the cells need to be “poisoned” first with Golgi inhibitors to prevent secretion, then permeabilized and fixed/“mummified.” The subsequent standard flow cytometric analysis does not make the distinction whether the analyte is indeed bound for secretion and thus is biologically active, or is retained in/on the cell. Right panel: In contrast, ELISPOT measures the actual secretory activity of pharmacologically untreated, living cells. The cells survive ELISPOT assays unharmed, and can be retested, phenotyped, expanded, cloned, or cryopreserved. Graphic artist: Gabor Pesthy.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Cytometry, Enzyme-linked Immunospot, Activity Assay, Clone Assay

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 4. The different functional avidities of antigen-reactive T cells. PBMC of an HLA-A2 positive subject were plated with different concentrations of individual A-2 restricted CEF peptides, as specified by the different symbols. A standard 24 h IFN-G ELISPOT assay was performed. Note how far apart the maximum stimulatory concentrations of the different peptides are.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Functional Assay, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Typical ELISPOT images of secreted cytokines from human PBMCs. Note the prominent inhibitory effect of oxidative stress on secretion of all cytokines, except IL-4 and IL5.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Analysis of a dual-analyte ELISPOT assay. QuantiHub 4.1 is capable of recognizing and detecting spots that have different colors as well as different shades of the same color within a large dynamic range.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Measured versus theoretical distribution of ELISPOT counts. IFN-G-transfected CHO cells were plated into an IFN-G ELISPOT assay at 15, 30, 60, and 120 cells per well, with 192 replicate wells per cell number, developed and counted with ImmonoSpot® Software. Distributions for spot counts are depicted for a Poisson distribution (dotted lines ) and a normal distribution (the solid line) with the means and a standard deviation corresponding to the square root of the means.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Enzyme-linked Immunospot, Transfection, Software, Standard Deviation

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Flow chart of ex vivo and cultured ELISPOT assay.

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Ex Vivo, Cell Culture, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 3. Example of cultured ELISPOT application: Correlation of memory responses with protection. Memory cell responses were determined by cultured IFN-G ELISPOT assay. Results of ELISPOT analysis are expressed as mean spot-forming cells (SFCs)/million cells. Cultured ELISPOT assays were performed before Mycobacterium bovis infection 14 weeks post-BCG vaccination and compared to outcome of infection with M. bovis at week 28 post-vaccination (animals were challenged with M. bovis at week 14 post- vaccination). Protection has been determined by bacterial load (log CFU/g tissue). Shown is the correlation of mean cultured ELISPOT responses and mean bacterial loads using data from unvaccinated cattle as well as cattle vaccinated with three different vaccines that induced various degrees of protection (from ref. 2).

Article Snippet: Commercially available ready-to-use ELISPOT assay kits (R&D Systems, Inc.) to study secretion of mouse TNF(EL410).

Techniques: Cell Culture, Enzyme-linked Immunospot, Infection, Vaccines

Journal: Theranostics

Article Title: Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1β

doi: 10.7150/thno.11432

Figure Lengend Snippet: Role of IL-1β in SLΔppGpp-mediated cancer therapy. (A) Protocols for co-treatment with an IL-1β blocking antibody (left) or recombinant IL-1β (right), plus PBS or SLΔppGpp. (B) CT26 cells were transplanted into mice. Mice then received an intravenous (i.v.) injection of PBS (black rectangles) or SLΔppGpp (black circles). IL-1β depletion: mice received an i.v. injection of anti-IL-1β-specific antibody (IL-1β Ab) 1 day before SLΔppGpp treatment (Day 1). The antibody was then injected twice a week for 2 weeks (open rectangles). Control mice received isotype (IgG) according to the same schedule (black triangles). Treatment with recombinant IL-1β (rIL-1β): mice received an intratumoral (i.t.) injection of rIL-1β on 5 dpi (open triangles), followed by another i.t injection every other day up until 11 dpi. Another group of tumor-bearing mice received four i.t injections of rIL-1β alone (open circles). Data represent the mean ± SD. Results from at least three individual experiments are shown. * P < 0.05, ** P < 0.005, and *** P < 0.001 vs. control at Day 12 and vs. SLΔppGpp at Day 18. (C) Photographs of representative animals in each group were taken before (0 dpi) and after treatment (5 and 16 dpi).

Article Snippet: The Salmonellae and TNF-α combination therapy groups received an intratumoral injection of recombinant TNF-α (410-MT/CF; 0.25 μg in PBS; R&D Systems) every 2 days starting at 5 dpi and continuing until 11 dpi.

Techniques: Blocking Assay, Recombinant, Injection, Control