human soluble tnfr1 protein  (R&D Systems)

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: TNFR1 is selectively sequestered into SC EVs. (a) SC EVs isolated by UC from conditioned media (CM) of primary cultured SCs were analyzed by NTA and had an average particle size of 153 ± 2 nm; n = 3 independent experiments. (b) Transmission electron microscopy (TEM) using negative staining of EVs. Note large crescent shaped particles and smaller particles. Scale bar, 200 nm. (c) Immunoblot analysis of whole SC lysates (SC-L) and SC-derived EVs to detect the exosome biomarkers, Flotillin-1, TSG101, CD9, ALIX, and CD81. GM130 is a Golgi biomarker not found in exosomes. (d) Immunoblot analysis to detect the SC biomarkers, p75NTR and myelin protein zero, P0, and non-myelinating marker, GFAP. Images represent n = 3–5 independent experiments. (e) TNFα receptors, TNFR1 (55-kDa) and TNFR2 (65-kDa), and P0 in extracts of SCs cultured in complete medium (SC-L1) or in DMEM with 10% FBS depleted of EVs (SC-L2), in SC-EVs, and in bone marrow derived macrophages (BMDMs) (2 μg/lane) were determined by immunoblot analysis. (f) Immunoblot of TNFR1 levels in EVs derived from SCs treated with and without TNFα. TSG101 (44-kDa) shows load control in cells and presence in EVs. (g) Quantification of TNFR1 levels in SC EV immunoblots (2 μg). Data are expressed as mean ± SEM; (n = 4–5/group)

Article Snippet: Different concentrations of recombinant human soluble TNFR1 protein (28-kDa; R&D Systems, 636-R1) were run on the same blots to generate a standard curve.

Techniques: Isolation, Cell Culture, Transmission Assay, Electron Microscopy, Negative Staining, Western Blot, Derivative Assay, Biomarker Discovery, Marker, Control

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: TNFR1 is expressed on the surface of SC EVs and binds TNFα in vitro. (a) Representative dot blot of non-permeabilized SC EVs (0–2.5 μg) probed with anti-TNFR1 antibody (n = 3 independent blots). (b) Standard curve showing recombinant soluble TNFR1 (28-kDa) and two representative SC EV samples (1 μg/lane). (c) Quantification of the TNFR1 copy number based on the TNFR1 standard curve, NTA and BCA analysis of six independent EV preparations. Data are expressed as the mean ± SEM. (d) Representative immunoblot of TNFα treated with the crosslinker, BS3 (+), or with vehicle (−) for the indicated times at 37°C. (e) Representative immunoblot of SC EVs, SC EV + TNFα and TNFα alone treated with BS3. Note the high molecular mass band in sample of EVs plus TNFα, compared with TNFα alone. (f) Dot blot of four independent SC EV preparations (EV1–4; 1 μg) incubated with or without biotinylated TNFα and probed with S-HRP

Article Snippet: Different concentrations of recombinant human soluble TNFR1 protein (28-kDa; R&D Systems, 636-R1) were run on the same blots to generate a standard curve.

Techniques: In Vitro, Dot Blot, Recombinant, Western Blot, Incubation

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: SC EV TNFR1 functions as a decoy by binding TNFα and inhibiting its association with cells. (a) Representative IF microscopy images identifying cell associated TNFα (red) and SC nuclei with DAPI (blue) in non-permeabilized SCs. Primary SCs were treated with TNFα (1 nM) in the presence or absence of SC EVs (5 μg) for 1 h and fixed in 4% paraformaldehyde. Scale bar, 50 μm. (b) Immunoblot of TNFα in SC extracts after incubating SCs with TNFα in the presence or absence of SC EVs (5 μg) for 1 h. Membranes were re-probed with GAPDH as a loading control (lower panel). (c) Densitometry analysis showing SC EV-induced reductions in cell-associated TNFα, standardized to the loading control (mean ± SEM; n = 3/group; *p < .05 using a one-way ANOVA and a Tukey’s post hoc test). (d) Immunoblot analysis identifying TNFα (1 nM) in SC CM after incubating SCs with TNFα in the presence or absence of SC EV (5 μg) for 1 h

Article Snippet: Different concentrations of recombinant human soluble TNFR1 protein (28-kDa; R&D Systems, 636-R1) were run on the same blots to generate a standard curve.

Techniques: Binding Assay, Microscopy, Western Blot, Control

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: SC EVs attenuate TNFα-induced p38 MAPK activation in vitro. (a) Representative immunoblot showing activation of p38 MAPK (P-p38) by TNFα (0.5 nM) and the effects of SC EVs (10 μg), when added simultaneously. Membranes were re-probed for total p38 MAPK (T-p38) as a loading control. Equal amounts of protein extracts (20 μg) were loaded into each lane. (b) Densitometry analysis was performed to determine the relative level of P-p38 standardized against T-p38 (mean ± SEM; n = 10 independent experiments, **p < .01, vehicle versus TNFα; **p < .01, TNFα versus SC EV using a one-way ANOVA with a Tukey’s post hoc test). (c) Representative immunoblot showing that TNFα (0.5 nM) activates p38 MAPK and that activation is not blocked by two concentrations of pre-incubated TNFR1-neutralizing antibody. (d) Immunoblot analysis of phospho-p38 MAPK in response to TNFα in the presence or absence of SC EVs. SC EVs were added to cultures together with TNFR1 neutralizing antibody (TNFR1 Ab; 2.5 or 10 μg) as indicated. The blot is representative of three independent experiments. (e) Immunoblot analysis of TNFR1 in pellet and supernatant of SC EVs treated with TACE/ADAM17 (20 or 80 nM). TSG101, an EV biomarker is identified in the pellet. Equal amounts of protein extracts (12 μg) were loaded into each lane. (f) Immunoblot analysis of TNFR1 in control SC EVs and TACE-treated SC EVs. TSG101 serves as a loading control. (g) Representative immunoblot showing activation of p38 MAPK by TNFα (0.5 nM) and the effects of independent preparations of TACE-EVs (10 μg/ml). Membranes were re-probed for T-p38 as a loading control. Equal amounts of protein extracts (20 μg) were loaded into each lane. (h) Densitometry analysis was performed to determine the relative level of P-p38 standardized against T-p38 (mean ± SEM; n = 6 independent experiments, ***p < .001, vehicle versus TNFα; ***p < .001 vehicle versus TNFα + TACE-EV; p = .997, n.s. TNFα versus TACE-EV using a one-way ANOVA with a Tukey’s post hoc test)

Article Snippet: Different concentrations of recombinant human soluble TNFR1 protein (28-kDa; R&D Systems, 636-R1) were run on the same blots to generate a standard curve.

Techniques: Activation Assay, In Vitro, Western Blot, Control, Incubation, Biomarker Discovery

Journal: Glia

Article Title: Tumor necrosis factor receptor-1 is selectively sequestered into Schwann cell extracellular vesicles where it functions as a TNFα decoy

doi: 10.1002/glia.24098

Figure Lengend Snippet: TNFR2 mediates TNFα induced P38 MAPK pathway in cultured SCs. (a) RT-qPCR analysis of TNFR2 mRNA after transfection with TNFR2 siRNA for 48 h. Data are presented as mean ± SEM; n = 4 independent experiments, *p < .05 using a t test. (b) Representative immunoblot analysis of phospho-p-38 MAPK (P-p38) that is dose-dependently increased by TNFα (8 min) in NTC cells, but not in cells transfected with TNFR2-specific siRNA (n = 3). (c) RT-qPCR analysis of TNFR1 mRNA after TNFR2 siRNA transfection for 48 h. Data are presented as mean ± SEM; n = 6 independent experiments, n.s. using a t test. (d) Representative immunoblot analysis of P-p38 after TNFα (0.5 nM) stimulation for 8 min in SCs treated dose-dependently with neutralizing anti-TNFR2 antibody (0–8 μg) or IgG control (8 μg)

Article Snippet: Different concentrations of recombinant human soluble TNFR1 protein (28-kDa; R&D Systems, 636-R1) were run on the same blots to generate a standard curve.

Techniques: Cell Culture, Quantitative RT-PCR, Transfection, Western Blot, Control

Journal: Cancers

Article Title: Inflammation-Driven Regulation of PD-L1 and PD-L2, and Their Cross-Interactions with Protective Soluble TNFα Receptors in Human Triple-Negative Breast Cancer

doi: 10.3390/cancers14143513

Figure Lengend Snippet: Recombinant and cell-derived sTNFR1 and sTNFR2 have a protective effect against TNFα-induced expression of pro-metastatic chemokines in MDA-MB-231 cells. MDA-MB-231 cells (MDA) were stimulated by TNFα (0.5 ng/mL) that was pre-incubated with rsTNFR1 (150 ng/mL), rsTNFR2 (500 ng/mL), rsTNFR1 + rsTNFR2 (concentrations as before) or their vehicle. When indicated, the cells were cultured prior to TNFα stimulation with TAPI-0 (5 µg/mL) or its vehicle for 3 h, as well as during cytokine stimulation (TAPI-0 did not affect tumor cell growth). The concentrations of rsTNFR1 and rsTNFR2 were selected as described in . CM were collected following 24 h stimulation, and CXCL8 ( A ) and CXCL1 ( B ) levels were determined by ELISA. A representative experiment of n = 3 is presented. *** p < 0.001, ** p < 0.01, * p < 0.05. # p < 0.1. NS, not significant. Black asterisks denote the differences in chemokine levels between TNFα-stimulated cells and vehicle-treated cells. Orange asterisks denote the differences in chemokine levels between TAPI-0-treated cells and cells treated by its vehicle. Statistical analyses were performed as described in .

Article Snippet: To determine the impact of recombinant soluble (rs) TNFR1 and rsTNFR2 on induction of TNFα-induced chemokine production, rsTNFR1 (150 ng/mL, #636-R1, R&D Systems), rsTNFR2 (500 ng/mL, #1089-R2, R&D Systems), both of them together or a vehicle control were incubated with TNFα (0.5 ng/mL, #300-01A, PeproTech) for 60 min at room temperature.

Techniques: Recombinant, Derivative Assay, Expressing, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Inflammation-Driven Regulation of PD-L1 and PD-L2, and Their Cross-Interactions with Protective Soluble TNFα Receptors in Human Triple-Negative Breast Cancer

doi: 10.3390/cancers14143513

Figure Lengend Snippet: Recombinant and cell-derived sTNFR1 and sTNFR2 have a protective effect against TNFα-induced expression of pro-metastatic chemokines in BT-549 cells. BT-549 cells (BT) were stimulated by TNFα (0.25–0.5 ng/mL) that was pre-incubated with rsTNFR1 (150 ng/mL), rsTNFR2 (500 ng/mL), rsTNFR1 + rsTNFR2 (concentrations as before) or their vehicle. When indicated, the cells were cultured prior to TNFα stimulation with TAPI-0 (5 µg/mL) or its vehicle for 3 h, as well as during cytokine stimulation (TAPI-0 did not affect tumor cell growth). The concentrations of rsTNFR1 and rsTNFR2 were selected as described in . CM were collected following 24 h stimulation, and CXCL8 ( A ), CXCL1 ( B ) and CCL5 ( C ) levels were determined by ELISA. A representative experiment of n = 3 is presented. *** p < 0.001, ** p < 0.01, * p < 0.05, # p < 0.1. NS, not significant. Black asterisks denote the differences in chemokine levels between TNFα-stimulated cells and vehicle-treated cells. Orange asterisks denote the differences in chemokine levels between TAPI-0-treated cells and cells treated by its vehicle. Statistical analyses were performed as described in .

Article Snippet: To determine the impact of recombinant soluble (rs) TNFR1 and rsTNFR2 on induction of TNFα-induced chemokine production, rsTNFR1 (150 ng/mL, #636-R1, R&D Systems), rsTNFR2 (500 ng/mL, #1089-R2, R&D Systems), both of them together or a vehicle control were incubated with TNFα (0.5 ng/mL, #300-01A, PeproTech) for 60 min at room temperature.

Techniques: Recombinant, Derivative Assay, Expressing, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay