sil 6rα  (R&D Systems)

 
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    Name:
    Recombinant Mouse IL 6 R alpha aa 20 357 Protein
    Description:
    The Recombinant Mouse IL 6 R alpha aa 20 357 Protein from R D Systems is derived from NS0 The Recombinant Mouse IL 6 R alpha aa 20 357 Protein has been validated for the following applications Binding Activity
    Catalog Number:
    1830-SR-025
    Price:
    329
    Category:
    Proteins and Enzymes
    Source:
    NS0-derived Recombinant Mouse IL-6 R alpha (aa 20-357) Protein
    Applications:
    Binding Activity
    Purity:
    >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie« Blue Staining.
    Conjugate:
    Unconjugated
    Size:
    25 ug
    Buy from Supplier


    Structured Review

    R&D Systems sil 6rα
    M10 intervention of the IL-6 signaling pathway in RA FLS (A) Western blotting determination of Bcl-2 levels in RA PBMC and FLS pretreated with M10 and cultured with <t>IL-6/sIL-6Rα</t> (100 ng/mL) for 72 h. (B) Western blot detection of STAT3 phosphorylation in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Western blot detection of STAT3 phosphorylation in normal PBMC, RA PBMC, RA SFMC, and RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 30 min. (D) FACS detection of the effect of M10 on STAT3 phosphorylation in normal PBMC, RA PBMC, and RA SFMC.
    The Recombinant Mouse IL 6 R alpha aa 20 357 Protein from R D Systems is derived from NS0 The Recombinant Mouse IL 6 R alpha aa 20 357 Protein has been validated for the following applications Binding Activity
    https://www.bioz.com/result/sil 6rα/product/R&D Systems
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sil 6rα - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Regulatory effect of anti-gp130 functional mAb on IL-6 mediated RANKL and Wnt5a expression through JAK-STAT3 signaling pathway in FLS"

    Article Title: Regulatory effect of anti-gp130 functional mAb on IL-6 mediated RANKL and Wnt5a expression through JAK-STAT3 signaling pathway in FLS

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23917

    M10 intervention of the IL-6 signaling pathway in RA FLS (A) Western blotting determination of Bcl-2 levels in RA PBMC and FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 72 h. (B) Western blot detection of STAT3 phosphorylation in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Western blot detection of STAT3 phosphorylation in normal PBMC, RA PBMC, RA SFMC, and RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 30 min. (D) FACS detection of the effect of M10 on STAT3 phosphorylation in normal PBMC, RA PBMC, and RA SFMC.
    Figure Legend Snippet: M10 intervention of the IL-6 signaling pathway in RA FLS (A) Western blotting determination of Bcl-2 levels in RA PBMC and FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 72 h. (B) Western blot detection of STAT3 phosphorylation in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Western blot detection of STAT3 phosphorylation in normal PBMC, RA PBMC, RA SFMC, and RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 30 min. (D) FACS detection of the effect of M10 on STAT3 phosphorylation in normal PBMC, RA PBMC, and RA SFMC.

    Techniques Used: Western Blot, Cell Culture, FACS

    Changes in IL-6/sIL-6Rα–induced RANKL and WNT5A expression after M10 pretreatment of RA FLS (A) Western blot detection of WNT5A and RANKL expression in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (B) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS pretreated with M10, and then cultured with IL-6/sIL-6Rα for 72 h. Data are normalized to β-actin and reported in relative expression units. (D) Immunostaining of RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα for 72 h (×200 magnification). Figures are representative of three independent experiments. (E) The number of RANKL- and WNT5A-positive staining cells.
    Figure Legend Snippet: Changes in IL-6/sIL-6Rα–induced RANKL and WNT5A expression after M10 pretreatment of RA FLS (A) Western blot detection of WNT5A and RANKL expression in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (B) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS pretreated with M10, and then cultured with IL-6/sIL-6Rα for 72 h. Data are normalized to β-actin and reported in relative expression units. (D) Immunostaining of RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα for 72 h (×200 magnification). Figures are representative of three independent experiments. (E) The number of RANKL- and WNT5A-positive staining cells.

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Immunostaining, Staining

    Schematic representation of the IL-6/sIL-6Rα/gp130 signaling pathways in RA FLS RA FLS release IL-6, which then interacts with IL-6Rα and gp130, forming a hexameric signaling complex and subsequently activating the JAK–STAT pathway and initiating RANKL, WNT5A, and Bcl-2 production.
    Figure Legend Snippet: Schematic representation of the IL-6/sIL-6Rα/gp130 signaling pathways in RA FLS RA FLS release IL-6, which then interacts with IL-6Rα and gp130, forming a hexameric signaling complex and subsequently activating the JAK–STAT pathway and initiating RANKL, WNT5A, and Bcl-2 production.

    Techniques Used:

    Analysis of IL-6/IL-6Rα/gp130 expression levels and RANKL in patients with RA (A) IL-6 levels in RA SF and in the control, OA, and RA serum. (B, C) sIL-6Rα and sgp130 levels in the control, OA, and RA serum and in the OA and RA SF. (D) Analysis of the sIL-6Rα and sgp130 ratio. (E) ELISA determination of soluble RANKL concentrations in RA and OA serum and SF. (F) Detection of MMP3 and TIMP1 in RA or OA serum and SF. (G) Western blot detection of RANKL expression in the bone tissues of patients with RA and the controls. (H) Bcl-2 levels in RA SF and in control, OA, and RA serum. (I) gp130 expression in the cells from the controls and patients with RA after 10-day induction with RANKL and GM-CSF; the percentage of gp130-positive cells was detected.
    Figure Legend Snippet: Analysis of IL-6/IL-6Rα/gp130 expression levels and RANKL in patients with RA (A) IL-6 levels in RA SF and in the control, OA, and RA serum. (B, C) sIL-6Rα and sgp130 levels in the control, OA, and RA serum and in the OA and RA SF. (D) Analysis of the sIL-6Rα and sgp130 ratio. (E) ELISA determination of soluble RANKL concentrations in RA and OA serum and SF. (F) Detection of MMP3 and TIMP1 in RA or OA serum and SF. (G) Western blot detection of RANKL expression in the bone tissues of patients with RA and the controls. (H) Bcl-2 levels in RA SF and in control, OA, and RA serum. (I) gp130 expression in the cells from the controls and patients with RA after 10-day induction with RANKL and GM-CSF; the percentage of gp130-positive cells was detected.

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

    2) Product Images from "IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis"

    Article Title: IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.008074

    IL-6 treatment stimulates bone formation in vivo but requires the addition of sIL-6R. A , schematic diagram. B , calvarial thickness in 6-week-old male C57BL/6 mice with vehicle (saline), hIL-6 alone (0.2 μg/day), hIL-6 (0.2 μg/day) with sIL-6rα (0.1 μg/day), hyper-IL-6 (0.2 μg/day), or murine OSM (0.2 μg/day) by subcutaneous calvarial injections. Data are shown as individual data points for each animal with mean ± S.E. ( error bars ); *, p
    Figure Legend Snippet: IL-6 treatment stimulates bone formation in vivo but requires the addition of sIL-6R. A , schematic diagram. B , calvarial thickness in 6-week-old male C57BL/6 mice with vehicle (saline), hIL-6 alone (0.2 μg/day), hIL-6 (0.2 μg/day) with sIL-6rα (0.1 μg/day), hyper-IL-6 (0.2 μg/day), or murine OSM (0.2 μg/day) by subcutaneous calvarial injections. Data are shown as individual data points for each animal with mean ± S.E. ( error bars ); *, p

    Techniques Used: In Vivo, Mouse Assay

    3) Product Images from "IL-6 Signaling in Myelomonocytic Cells Is Not Crucial for the Development of IMQ-Induced Psoriasis"

    Article Title: IL-6 Signaling in Myelomonocytic Cells Is Not Crucial for the Development of IMQ-Induced Psoriasis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0151913

    Functionality of Il6ra Δmyel mice. (A) Flow cytometric analysis of IL-6Rα expression of different organs. CD4 + cells are pre-gated on living CD90.2 + cells. Neutrophils (Gr-1 hi F4/80 - ), monocytes (Gr-1 int F4/80 + ) and macrophages (Gr-1 - F4/80 + ) are pre-gated on living CD90.2 - /B220 - and CD11b + cells. Gray histograms represent IgG2b isotype control for the IL-6R staining. Numbers in upper right corner represent the mean Mean Fluorescent Intensity (MFI) values of Il6ra Δmyel or wt cells. Shown are representative histograms (n = 7 or 8 (3 independent experiments). (B) MFI of IL-6Rα expression pre-gated on CD4 + cells, neutrophils (Gr-1 hi F4/80 - ), monocytes (Gr-1 int F4/80 + ) or macrophages (Gr-1 - F4/80 + ). Data are shown as bar graphs with mean and SEM. *p ≤ 0,05 Significance was calculated using Mann Whitney test. (C) Quantitative RT-PCR from CD11b + MACS purified bone marrow cells for the IL-6Rα gene in wt and Il6ra Δmyel mice. Expression levels are shown relative to the housekeeping gene HPRT (n = 5). Data are shown as bar graphs with mean and SEM. * p ≤ 0,05 Significance was calculated using Mann Whitney test. (D) Serum concentrations of sIL-6Rα examined by ELISA in wt (n = 6) and Il6ra Δmyel (n = 10) mice at the age of 5 weeks to 5 months. Data are shown as bar graphs with mean and SEM. *** p ≤ 0,001 Significance was calculated using Mann Whitney test.
    Figure Legend Snippet: Functionality of Il6ra Δmyel mice. (A) Flow cytometric analysis of IL-6Rα expression of different organs. CD4 + cells are pre-gated on living CD90.2 + cells. Neutrophils (Gr-1 hi F4/80 - ), monocytes (Gr-1 int F4/80 + ) and macrophages (Gr-1 - F4/80 + ) are pre-gated on living CD90.2 - /B220 - and CD11b + cells. Gray histograms represent IgG2b isotype control for the IL-6R staining. Numbers in upper right corner represent the mean Mean Fluorescent Intensity (MFI) values of Il6ra Δmyel or wt cells. Shown are representative histograms (n = 7 or 8 (3 independent experiments). (B) MFI of IL-6Rα expression pre-gated on CD4 + cells, neutrophils (Gr-1 hi F4/80 - ), monocytes (Gr-1 int F4/80 + ) or macrophages (Gr-1 - F4/80 + ). Data are shown as bar graphs with mean and SEM. *p ≤ 0,05 Significance was calculated using Mann Whitney test. (C) Quantitative RT-PCR from CD11b + MACS purified bone marrow cells for the IL-6Rα gene in wt and Il6ra Δmyel mice. Expression levels are shown relative to the housekeeping gene HPRT (n = 5). Data are shown as bar graphs with mean and SEM. * p ≤ 0,05 Significance was calculated using Mann Whitney test. (D) Serum concentrations of sIL-6Rα examined by ELISA in wt (n = 6) and Il6ra Δmyel (n = 10) mice at the age of 5 weeks to 5 months. Data are shown as bar graphs with mean and SEM. *** p ≤ 0,001 Significance was calculated using Mann Whitney test.

    Techniques Used: Mouse Assay, Flow Cytometry, Expressing, Staining, MANN-WHITNEY, Quantitative RT-PCR, Magnetic Cell Separation, Purification, Enzyme-linked Immunosorbent Assay

    4) Product Images from "Associations between the IL-6-neutralizing sIL-6R-sgp130 buffer system and coronary artery disease in postmenopausal women"

    Article Title: Associations between the IL-6-neutralizing sIL-6R-sgp130 buffer system and coronary artery disease in postmenopausal women

    Journal: Annals of Translational Medicine

    doi: 10.21037/atm.2020.02.27

    Receiver operating characteristic (ROC) curve analysis to assess how well Gensini scores and parameters of the IL-6-neutralizing sIL-6R-sgp130 buffer system can predict coronary artery disease in postmenopausal women. AUC, area under the receiver operating characteristic curve; 95% CI, 95% confidence interval; IL-6, interleukin-6; sIL-6Rα, soluble interleukin-6 receptor α; sgp130, soluble glycoprotein 130; B/T ratio, binary/ternary complex ratio.
    Figure Legend Snippet: Receiver operating characteristic (ROC) curve analysis to assess how well Gensini scores and parameters of the IL-6-neutralizing sIL-6R-sgp130 buffer system can predict coronary artery disease in postmenopausal women. AUC, area under the receiver operating characteristic curve; 95% CI, 95% confidence interval; IL-6, interleukin-6; sIL-6Rα, soluble interleukin-6 receptor α; sgp130, soluble glycoprotein 130; B/T ratio, binary/ternary complex ratio.

    Techniques Used:

    5) Product Images from "STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation"

    Article Title: STAT-3 contributes to pulmonary fibrosis through epithelial injury and fibroblast-myofibroblast differentiation

    Journal: The FASEB Journal

    doi: 10.1096/fj.15-273953

    STAT-3 inhibition on lung fibroblasts. A ) Lung fibroblasts were plated, treated for 1 h with different inhibitors (C-188-9, 20 μM; SMAD2 and 3 inhibitor, 10 μM), and subsequently stimulated with TGF-β (10 ng/ml) and IL-6/sIL-6Rα
    Figure Legend Snippet: STAT-3 inhibition on lung fibroblasts. A ) Lung fibroblasts were plated, treated for 1 h with different inhibitors (C-188-9, 20 μM; SMAD2 and 3 inhibitor, 10 μM), and subsequently stimulated with TGF-β (10 ng/ml) and IL-6/sIL-6Rα

    Techniques Used: Inhibition

    Profibrotic mediators on different cells following STAT-3 inhibition or knockdown. MLE-12 was plated and subsequently stimulated with TGF-β (10 ng/ml), IL-6/sIL-6Rα complex (40 μg/ml), and C-188-9 inhibitor (20 μM); 24
    Figure Legend Snippet: Profibrotic mediators on different cells following STAT-3 inhibition or knockdown. MLE-12 was plated and subsequently stimulated with TGF-β (10 ng/ml), IL-6/sIL-6Rα complex (40 μg/ml), and C-188-9 inhibitor (20 μM); 24

    Techniques Used: Inhibition

    Myofibroblast differentiation on lung fibroblasts following STAT-3 inhibition. Primary lung fibroblasts were isolated, plated, and subsequently stimulated with TGF-β (10 ng/ml), IL-6/sIL-6Rα complex (40 μg/ml), and C-188-9 inhibitor
    Figure Legend Snippet: Myofibroblast differentiation on lung fibroblasts following STAT-3 inhibition. Primary lung fibroblasts were isolated, plated, and subsequently stimulated with TGF-β (10 ng/ml), IL-6/sIL-6Rα complex (40 μg/ml), and C-188-9 inhibitor

    Techniques Used: Inhibition, Isolation

    6) Product Images from "Anti-interleukin-6 receptor antibody treatment ameliorates postoperative adhesion formation"

    Article Title: Anti-interleukin-6 receptor antibody treatment ameliorates postoperative adhesion formation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54175-1

    Production of pro-fibrotic molecules by human neutrophils in response to proinflammatory cytokines. Expression of IL6 , TNF , and TGFB1 was determined in human neutrophils stimulated with TNF-α, IL-6, or TGF-β1 using qRT-PCR ( a ). Human mesothelial cells (MeT5A cells) were incubated with TNF-α, IL-6 plus soluble IL-6Rα (sIL-6Rα), TGF-β1, or IFN-γ, followed by measurement of IL6 ( b ), TNF ( b ), CXCL2 ( b ), TGFB1 ( b ), or COL1A1 expression by qRT-PCR ( c ). Three independent experiments were performed. Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Production of pro-fibrotic molecules by human neutrophils in response to proinflammatory cytokines. Expression of IL6 , TNF , and TGFB1 was determined in human neutrophils stimulated with TNF-α, IL-6, or TGF-β1 using qRT-PCR ( a ). Human mesothelial cells (MeT5A cells) were incubated with TNF-α, IL-6 plus soluble IL-6Rα (sIL-6Rα), TGF-β1, or IFN-γ, followed by measurement of IL6 ( b ), TNF ( b ), CXCL2 ( b ), TGFB1 ( b ), or COL1A1 expression by qRT-PCR ( c ). Three independent experiments were performed. Data are shown as mean ± SEM. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Incubation

    Anti-IL-6Rα antibody protection against postoperative adhesion formation. Wild-type mice were treated with various doses of anti-IL-6Rα antibody or isotype-matched antibody and subjected to cecum cauterization. ( a ) Adhesion scores were determined on day 7 following the procedure. Representative photos are shown. ( b,c ) Cecum lesions were sampled at the indicated time points for indicated protein staining ( b ) and for qRT-PCR analysis of indicated proinflammatory and pro-fibrotic mRNA transcripts ( c ). Each experimental group contained 3–5 mice, with two independent experiments performed. Representative photos are shown. Data at 0 day or hour postoperation indicated those in untreated control mice. Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Anti-IL-6Rα antibody protection against postoperative adhesion formation. Wild-type mice were treated with various doses of anti-IL-6Rα antibody or isotype-matched antibody and subjected to cecum cauterization. ( a ) Adhesion scores were determined on day 7 following the procedure. Representative photos are shown. ( b,c ) Cecum lesions were sampled at the indicated time points for indicated protein staining ( b ) and for qRT-PCR analysis of indicated proinflammatory and pro-fibrotic mRNA transcripts ( c ). Each experimental group contained 3–5 mice, with two independent experiments performed. Representative photos are shown. Data at 0 day or hour postoperation indicated those in untreated control mice. Data are shown as mean ± SEM. * p

    Techniques Used: Mouse Assay, Staining, Quantitative RT-PCR

    7) Product Images from "Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes"

    Article Title: Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00256

    IL-27p28 and IL-6 sequence homology and proposed model of IL-30 and IL-27 interactions with IL-27 receptor α/T cell cytokine receptor (WSX-1), glycoprotein 130 (gp130), and soluble IL-6 receptor α (sIL-6Rα). (A) Human IL-27p28 (accession number: NP_663634.2) and human IL-6 (accession number: NP_000591.1) sequences were aligned using the T-COFFEE-expresso program ( 32 ). α-helices A, B, C, and D are indicated by bold red letters and boxed sequences. Conserved Trp 197 on IL-27p28 and Trp 185 on IL-6 are indicated by the red arrow. [ (B) ; left] The p28 subunit contains three binding sites (site 1, site 2, and site 3). For the binding of the dimeric IL-27, it has been demonstrated that the p28 site 1 interacts with Epstein–Barr virus-induced gene 3 (EBI3), site 2 interacts with WSX-1, and site 3 interacts with gp130 ( 36 ). For the binding of IL-30 (IL-27p28) to the WSX-1 and gp130 receptor chains, sIL-6Rα may be required. Since sIL-6Rα interacts with site 1 on IL-6, and IL-6, and IL-30 share homology, the p28 site 2 may interact with sIL-6Rα to enable IL-30-mediated signaling. Furthermore, sIL-6Rα may serve as an alternative binding partner in the absence of EBI3 to induce IL-30 signaling. [ (B) ; right] IL-6 may compete with IL-30 for interaction with sIL-6Rα due to higher affinity for IL-6. As a result, in the presence of IL-6, both IL-30 and IL-27 signaling and downstream functions are inhibited.
    Figure Legend Snippet: IL-27p28 and IL-6 sequence homology and proposed model of IL-30 and IL-27 interactions with IL-27 receptor α/T cell cytokine receptor (WSX-1), glycoprotein 130 (gp130), and soluble IL-6 receptor α (sIL-6Rα). (A) Human IL-27p28 (accession number: NP_663634.2) and human IL-6 (accession number: NP_000591.1) sequences were aligned using the T-COFFEE-expresso program ( 32 ). α-helices A, B, C, and D are indicated by bold red letters and boxed sequences. Conserved Trp 197 on IL-27p28 and Trp 185 on IL-6 are indicated by the red arrow. [ (B) ; left] The p28 subunit contains three binding sites (site 1, site 2, and site 3). For the binding of the dimeric IL-27, it has been demonstrated that the p28 site 1 interacts with Epstein–Barr virus-induced gene 3 (EBI3), site 2 interacts with WSX-1, and site 3 interacts with gp130 ( 36 ). For the binding of IL-30 (IL-27p28) to the WSX-1 and gp130 receptor chains, sIL-6Rα may be required. Since sIL-6Rα interacts with site 1 on IL-6, and IL-6, and IL-30 share homology, the p28 site 2 may interact with sIL-6Rα to enable IL-30-mediated signaling. Furthermore, sIL-6Rα may serve as an alternative binding partner in the absence of EBI3 to induce IL-30 signaling. [ (B) ; right] IL-6 may compete with IL-30 for interaction with sIL-6Rα due to higher affinity for IL-6. As a result, in the presence of IL-6, both IL-30 and IL-27 signaling and downstream functions are inhibited.

    Techniques Used: Sequencing, Binding Assay

    Recombinant IL-6 inhibits IL-27- and IL-30-IP-10 production. (A) THP-1 cells and (B) primary human monocytes were pretreated with recombinant IL-6 (10 ng/mL) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of 13 different THP-1 experiments or 6 different monocyte donors. THP-1 cells and primary human monocytes were stimulated with or without recombinant IL-6 (10 ng/mL) for 30 min and then stained with IL-27 receptor α/T cell cytokine receptor (WSX-1) (C) , glycoprotein 130 (gp130) (D) , or anti-IL-6Rα (E) . Mann–Whitney U tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated THP-1 cells. Wilcoxon signed-rank matched pair tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated primary monocytes. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Recombinant IL-6 inhibits IL-27- and IL-30-IP-10 production. (A) THP-1 cells and (B) primary human monocytes were pretreated with recombinant IL-6 (10 ng/mL) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of 13 different THP-1 experiments or 6 different monocyte donors. THP-1 cells and primary human monocytes were stimulated with or without recombinant IL-6 (10 ng/mL) for 30 min and then stained with IL-27 receptor α/T cell cytokine receptor (WSX-1) (C) , glycoprotein 130 (gp130) (D) , or anti-IL-6Rα (E) . Mann–Whitney U tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated THP-1 cells. Wilcoxon signed-rank matched pair tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated primary monocytes. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

    Cleaved soluble IL-6 receptor α (sIL-6Rα) is involved IL-27 and IL-30 function. (A) THP-1 cells were cultured in medium (RPMI + 10% FBS) for 4, 8, 16, and 24 h. Endogenously produced sIL-6Rα was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of three different THP-1 experiments. (B) THP-1 cells and (C) primary human monocytes were treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h, and sIL-6Rα was measured in cell-free supernatants by ELISA. (D,E) Primary human monocytes were pretreated with BB-94 (25 µM) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. (D) sIL-6Rα production and (E) IP-10 production were measured in cell-free supernatants by ELISA. (F,G) Recombinant human IL-6Rα was preincubated with IL-27 or IL-30 for 2 h. THP-1 cells were pretreated with BB-94 (25 µM) for 1 h and then stimulated with preincubated IL-6Rα (2.5 ng/mL) with or without IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. Pretreatment with DMSO was used as a vehicle control for BB-94. (F) sIL-6Rα production, and (G) IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of at least three different THP-1 experiments or three different monocyte donors. Mann–Whitney U tests were used for statistical analyses between indicated groups (B,C) , between corresponding controls (DMSO) and BB-94 treatment (D,E) , or between pairs as indicated (F,G) , unless otherwise specified. Wilcoxon signed-rank matched pair tests were used for statistical analyses between medium or IL-30 control (DMSO) and BB-94 treatment (E) , between medium, IL-27, or IL-30 DMSO and DMSO + rhIL-6Rα treatment (F) , and between IL-27 or IL-30 BB-94 and BB-94 + rhIL-6Rα treatment (G) . ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Cleaved soluble IL-6 receptor α (sIL-6Rα) is involved IL-27 and IL-30 function. (A) THP-1 cells were cultured in medium (RPMI + 10% FBS) for 4, 8, 16, and 24 h. Endogenously produced sIL-6Rα was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of three different THP-1 experiments. (B) THP-1 cells and (C) primary human monocytes were treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h, and sIL-6Rα was measured in cell-free supernatants by ELISA. (D,E) Primary human monocytes were pretreated with BB-94 (25 µM) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. (D) sIL-6Rα production and (E) IP-10 production were measured in cell-free supernatants by ELISA. (F,G) Recombinant human IL-6Rα was preincubated with IL-27 or IL-30 for 2 h. THP-1 cells were pretreated with BB-94 (25 µM) for 1 h and then stimulated with preincubated IL-6Rα (2.5 ng/mL) with or without IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. Pretreatment with DMSO was used as a vehicle control for BB-94. (F) sIL-6Rα production, and (G) IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of at least three different THP-1 experiments or three different monocyte donors. Mann–Whitney U tests were used for statistical analyses between indicated groups (B,C) , between corresponding controls (DMSO) and BB-94 treatment (D,E) , or between pairs as indicated (F,G) , unless otherwise specified. Wilcoxon signed-rank matched pair tests were used for statistical analyses between medium or IL-30 control (DMSO) and BB-94 treatment (E) , between medium, IL-27, or IL-30 DMSO and DMSO + rhIL-6Rα treatment (F) , and between IL-27 or IL-30 BB-94 and BB-94 + rhIL-6Rα treatment (G) . ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Cell Culture, Produced, Enzyme-linked Immunosorbent Assay, Recombinant, MANN-WHITNEY

    8) Product Images from "Anti-interleukin-6 receptor antibody treatment ameliorates postoperative adhesion formation"

    Article Title: Anti-interleukin-6 receptor antibody treatment ameliorates postoperative adhesion formation

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54175-1

    Production of pro-fibrotic molecules by human neutrophils in response to proinflammatory cytokines. Expression of IL6 , TNF , and TGFB1 was determined in human neutrophils stimulated with TNF-α, IL-6, or TGF-β1 using qRT-PCR ( a ). Human mesothelial cells (MeT5A cells) were incubated with TNF-α, IL-6 plus soluble IL-6Rα (sIL-6Rα), TGF-β1, or IFN-γ, followed by measurement of IL6 ( b ), TNF ( b ), CXCL2 ( b ), TGFB1 ( b ), or COL1A1 expression by qRT-PCR ( c ). Three independent experiments were performed. Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Production of pro-fibrotic molecules by human neutrophils in response to proinflammatory cytokines. Expression of IL6 , TNF , and TGFB1 was determined in human neutrophils stimulated with TNF-α, IL-6, or TGF-β1 using qRT-PCR ( a ). Human mesothelial cells (MeT5A cells) were incubated with TNF-α, IL-6 plus soluble IL-6Rα (sIL-6Rα), TGF-β1, or IFN-γ, followed by measurement of IL6 ( b ), TNF ( b ), CXCL2 ( b ), TGFB1 ( b ), or COL1A1 expression by qRT-PCR ( c ). Three independent experiments were performed. Data are shown as mean ± SEM. * p

    Techniques Used: Expressing, Quantitative RT-PCR, Incubation

    Anti-IL-6Rα antibody protection against postoperative adhesion formation. Wild-type mice were treated with various doses of anti-IL-6Rα antibody or isotype-matched antibody and subjected to cecum cauterization. ( a ) Adhesion scores were determined on day 7 following the procedure. Representative photos are shown. ( b,c ) Cecum lesions were sampled at the indicated time points for indicated protein staining ( b ) and for qRT-PCR analysis of indicated proinflammatory and pro-fibrotic mRNA transcripts ( c ). Each experimental group contained 3–5 mice, with two independent experiments performed. Representative photos are shown. Data at 0 day or hour postoperation indicated those in untreated control mice. Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Anti-IL-6Rα antibody protection against postoperative adhesion formation. Wild-type mice were treated with various doses of anti-IL-6Rα antibody or isotype-matched antibody and subjected to cecum cauterization. ( a ) Adhesion scores were determined on day 7 following the procedure. Representative photos are shown. ( b,c ) Cecum lesions were sampled at the indicated time points for indicated protein staining ( b ) and for qRT-PCR analysis of indicated proinflammatory and pro-fibrotic mRNA transcripts ( c ). Each experimental group contained 3–5 mice, with two independent experiments performed. Representative photos are shown. Data at 0 day or hour postoperation indicated those in untreated control mice. Data are shown as mean ± SEM. * p

    Techniques Used: Mouse Assay, Staining, Quantitative RT-PCR

    9) Product Images from "Blockade of IL-6 Trans Signaling Attenuates Pulmonary Fibrosis"

    Article Title: Blockade of IL-6 Trans Signaling Attenuates Pulmonary Fibrosis

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1302470

    Effect of IL-6 trans signaling on proliferation rates and extracellular matrix protein production in control and IPF fibroblasts. Control and IPF fibroblasts were serum-starved for 24 h and then stimulated for 48 h with IL-6 alone or IL-6 and sIL-6Rα.
    Figure Legend Snippet: Effect of IL-6 trans signaling on proliferation rates and extracellular matrix protein production in control and IPF fibroblasts. Control and IPF fibroblasts were serum-starved for 24 h and then stimulated for 48 h with IL-6 alone or IL-6 and sIL-6Rα.

    Techniques Used:

    Pharmacologic neutralization and siRNA-mediated silencing of ADAM17 activity in macrophages. ( A ) Western blot analysis of mIL-6Rα and arginase 1 expression in bone marrow–derived macrophages stimulated with IL-4 and IL-13. ( B ) ELISA measurement
    Figure Legend Snippet: Pharmacologic neutralization and siRNA-mediated silencing of ADAM17 activity in macrophages. ( A ) Western blot analysis of mIL-6Rα and arginase 1 expression in bone marrow–derived macrophages stimulated with IL-4 and IL-13. ( B ) ELISA measurement

    Techniques Used: Neutralization, Activity Assay, Western Blot, Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay

    Model of IL-6 trans signaling in pulmonary fibrosis. In fibrotic lungs, elevated ADAM17 expression in M 2 macrophages leads to cleavage of mIL-6Rα to produce sIL-6Rα. sIL-6Rα binds IL-6. The IL-6/sIL-6Rα complex can then
    Figure Legend Snippet: Model of IL-6 trans signaling in pulmonary fibrosis. In fibrotic lungs, elevated ADAM17 expression in M 2 macrophages leads to cleavage of mIL-6Rα to produce sIL-6Rα. sIL-6Rα binds IL-6. The IL-6/sIL-6Rα complex can then

    Techniques Used: Expressing

    sIL-6Rα in IPF and a chronic bleomycin mouse model of pulmonary fibrosis. sIL-6Rα expression was assessed in humans and mice with pulmonary fibrosis. ( A and B ) ELISA measurement and Western blot analysis of sIL-6Rα in lung lysates
    Figure Legend Snippet: sIL-6Rα in IPF and a chronic bleomycin mouse model of pulmonary fibrosis. sIL-6Rα expression was assessed in humans and mice with pulmonary fibrosis. ( A and B ) ELISA measurement and Western blot analysis of sIL-6Rα in lung lysates

    Techniques Used: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Western Blot

    10) Product Images from "Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes"

    Article Title: Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.00256

    Recombinant IL-6 inhibits IL-27- and IL-30-IP-10 production. (A) THP-1 cells and (B) primary human monocytes were pretreated with recombinant IL-6 (10 ng/mL) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of 13 different THP-1 experiments or 6 different monocyte donors. THP-1 cells and primary human monocytes were stimulated with or without recombinant IL-6 (10 ng/mL) for 30 min and then stained with IL-27 receptor α/T cell cytokine receptor (WSX-1) (C) , glycoprotein 130 (gp130) (D) , or anti-IL-6Rα (E) . Mann–Whitney U tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated THP-1 cells. Wilcoxon signed-rank matched pair tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated primary monocytes. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Recombinant IL-6 inhibits IL-27- and IL-30-IP-10 production. (A) THP-1 cells and (B) primary human monocytes were pretreated with recombinant IL-6 (10 ng/mL) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of 13 different THP-1 experiments or 6 different monocyte donors. THP-1 cells and primary human monocytes were stimulated with or without recombinant IL-6 (10 ng/mL) for 30 min and then stained with IL-27 receptor α/T cell cytokine receptor (WSX-1) (C) , glycoprotein 130 (gp130) (D) , or anti-IL-6Rα (E) . Mann–Whitney U tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated THP-1 cells. Wilcoxon signed-rank matched pair tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated primary monocytes. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

    Cleaved soluble IL-6 receptor α (sIL-6Rα) is involved IL-27 and IL-30 function. (A) THP-1 cells were cultured in medium (RPMI + 10% FBS) for 4, 8, 16, and 24 h. Endogenously produced sIL-6Rα was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of three different THP-1 experiments. (B) THP-1 cells and (C) primary human monocytes were treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h, and sIL-6Rα was measured in cell-free supernatants by ELISA. (D,E) Primary human monocytes were pretreated with BB-94 (25 µM) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. (D) sIL-6Rα production and (E) IP-10 production were measured in cell-free supernatants by ELISA. (F,G) Recombinant human IL-6Rα was preincubated with IL-27 or IL-30 for 2 h. THP-1 cells were pretreated with BB-94 (25 µM) for 1 h and then stimulated with preincubated IL-6Rα (2.5 ng/mL) with or without IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. Pretreatment with DMSO was used as a vehicle control for BB-94. (F) sIL-6Rα production, and (G) IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of at least three different THP-1 experiments or three different monocyte donors. Mann–Whitney U tests were used for statistical analyses between indicated groups (B,C) , between corresponding controls (DMSO) and BB-94 treatment (D,E) , or between pairs as indicated (F,G) , unless otherwise specified. Wilcoxon signed-rank matched pair tests were used for statistical analyses between medium or IL-30 control (DMSO) and BB-94 treatment (E) , between medium, IL-27, or IL-30 DMSO and DMSO + rhIL-6Rα treatment (F) , and between IL-27 or IL-30 BB-94 and BB-94 + rhIL-6Rα treatment (G) . ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
    Figure Legend Snippet: Cleaved soluble IL-6 receptor α (sIL-6Rα) is involved IL-27 and IL-30 function. (A) THP-1 cells were cultured in medium (RPMI + 10% FBS) for 4, 8, 16, and 24 h. Endogenously produced sIL-6Rα was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of three different THP-1 experiments. (B) THP-1 cells and (C) primary human monocytes were treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h, and sIL-6Rα was measured in cell-free supernatants by ELISA. (D,E) Primary human monocytes were pretreated with BB-94 (25 µM) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. (D) sIL-6Rα production and (E) IP-10 production were measured in cell-free supernatants by ELISA. (F,G) Recombinant human IL-6Rα was preincubated with IL-27 or IL-30 for 2 h. THP-1 cells were pretreated with BB-94 (25 µM) for 1 h and then stimulated with preincubated IL-6Rα (2.5 ng/mL) with or without IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. Pretreatment with DMSO was used as a vehicle control for BB-94. (F) sIL-6Rα production, and (G) IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of at least three different THP-1 experiments or three different monocyte donors. Mann–Whitney U tests were used for statistical analyses between indicated groups (B,C) , between corresponding controls (DMSO) and BB-94 treatment (D,E) , or between pairs as indicated (F,G) , unless otherwise specified. Wilcoxon signed-rank matched pair tests were used for statistical analyses between medium or IL-30 control (DMSO) and BB-94 treatment (E) , between medium, IL-27, or IL-30 DMSO and DMSO + rhIL-6Rα treatment (F) , and between IL-27 or IL-30 BB-94 and BB-94 + rhIL-6Rα treatment (G) . ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Techniques Used: Cell Culture, Produced, Enzyme-linked Immunosorbent Assay, Recombinant, MANN-WHITNEY

    11) Product Images from "Temporal and tissue-specific requirements for T-lymphocyte IL-6 signalling in obesity-associated inflammation and insulin resistance"

    Article Title: Temporal and tissue-specific requirements for T-lymphocyte IL-6 signalling in obesity-associated inflammation and insulin resistance

    Journal: Nature Communications

    doi: 10.1038/ncomms14803

    Inflammatory profile of liver and EWAT (8-week HFD). ( a ) Liver ( n =10 versus 12) and ( b ) EWAT ( n =11) gene expression profile. Activity of c-Jun kinase (JNK) in ( c ) liver and ( d ) EWAT with the measurement of pS63 c-Jun by immunoblotting (representative immunoblots of 2 blots) and quantification ( n =8); relative unit (RU) to that of IL-6Rα f/f (Control). Flow cytometry analyses of ( e ) liver and ( f ) EWAT composition of total T cells, CD8+ and CD4+ T cells, T helper cells (Th1, Th17 and Th2) and CD25+ regulatory T cells (Treg), presented as percentage of live immune cells (PLICs) positive for CD45 ( n =3 analysed samples pooled from n =4–8 animals per sample). Flow cytometry analyses of ( g ) liver and ( h ) EWAT composition of total CD11c+ myeloid cells, F4/80+ myeloid cells, total macrophages (MΦ), CD11c+ MΦ and F4/80+ dendritic cells (DCs), presented as PLICs ( n =3 analysed samples pooled from n =4–8 animals per sample). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P
    Figure Legend Snippet: Inflammatory profile of liver and EWAT (8-week HFD). ( a ) Liver ( n =10 versus 12) and ( b ) EWAT ( n =11) gene expression profile. Activity of c-Jun kinase (JNK) in ( c ) liver and ( d ) EWAT with the measurement of pS63 c-Jun by immunoblotting (representative immunoblots of 2 blots) and quantification ( n =8); relative unit (RU) to that of IL-6Rα f/f (Control). Flow cytometry analyses of ( e ) liver and ( f ) EWAT composition of total T cells, CD8+ and CD4+ T cells, T helper cells (Th1, Th17 and Th2) and CD25+ regulatory T cells (Treg), presented as percentage of live immune cells (PLICs) positive for CD45 ( n =3 analysed samples pooled from n =4–8 animals per sample). Flow cytometry analyses of ( g ) liver and ( h ) EWAT composition of total CD11c+ myeloid cells, F4/80+ myeloid cells, total macrophages (MΦ), CD11c+ MΦ and F4/80+ dendritic cells (DCs), presented as PLICs ( n =3 analysed samples pooled from n =4–8 animals per sample). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P

    Techniques Used: Expressing, Activity Assay, Western Blot, Flow Cytometry, Cytometry, Two Tailed Test

    Augmented IL-6 signalling via sIL-6Rα after prolonged HFD feeding normalizes T cell functions in IL-6Rα T-KO (8- and 16-week HFD). ( a ) Compiled representative histograms and ( b ) quantification (MFI) of flow cytometry analyses of stimulated pY701 STAT-1 and pY705 STAT-3 with control (unstimulated), IL-6 (70 ng ml −1 ), IL-6—IL-6Rα complex (200 ng ml −1 ) and soluble IL-6Rα alone (130 ng ml −1 ) in enriched splenic T cell isolation from random-fed IL-6Rα f/f and IL-6Rα T-KO animals at 8 and 16 weeks of HFD feeding ( n =10–12 versus 10–12). ( c ) Flow cytometry analyses of T cell chemotaxis with control (no treatment), IL-6 (70 ng ml −1 ) and IL-6—IL-6Rα complex (200 ng ml −1 ), presented as percentage of cells pre-chemotaxis (PPC); enriched splenic T cells isolated from random-fed IL-6Rα f/f and IL-6Rα T-KO animals at 8 weeks ( n =6 versus 4) and 16 weeks ( n =4 versus 7) of HFD feeding. Two-way analysis of variance (ANOVA) with multiple analysis used for statistical analyses (* P
    Figure Legend Snippet: Augmented IL-6 signalling via sIL-6Rα after prolonged HFD feeding normalizes T cell functions in IL-6Rα T-KO (8- and 16-week HFD). ( a ) Compiled representative histograms and ( b ) quantification (MFI) of flow cytometry analyses of stimulated pY701 STAT-1 and pY705 STAT-3 with control (unstimulated), IL-6 (70 ng ml −1 ), IL-6—IL-6Rα complex (200 ng ml −1 ) and soluble IL-6Rα alone (130 ng ml −1 ) in enriched splenic T cell isolation from random-fed IL-6Rα f/f and IL-6Rα T-KO animals at 8 and 16 weeks of HFD feeding ( n =10–12 versus 10–12). ( c ) Flow cytometry analyses of T cell chemotaxis with control (no treatment), IL-6 (70 ng ml −1 ) and IL-6—IL-6Rα complex (200 ng ml −1 ), presented as percentage of cells pre-chemotaxis (PPC); enriched splenic T cells isolated from random-fed IL-6Rα f/f and IL-6Rα T-KO animals at 8 weeks ( n =6 versus 4) and 16 weeks ( n =4 versus 7) of HFD feeding. Two-way analysis of variance (ANOVA) with multiple analysis used for statistical analyses (* P

    Techniques Used: Flow Cytometry, Cytometry, Cell Isolation, Chemotaxis Assay, Isolation

    Improved glucose homeostasis and lipid metabolism in IL-6Rα T-KO (8-week HFD). ( a ) Blood glucose (BG) and glucose infusion rate (GIR) during hyperinsulinaemic-euglycaemic (HIEG) clamp experiments ( n =9); two-way analysis of variance (ANOVA) was performed on data from the green-shaded steady state (130–180 min). ( b ) Rate of hepatic glucose production (HGP) at basal and during steady state of HIEG clamp ( n =9). ( c ) Rate of glucose uptake (GU) during steady state of HIEG clamp ( n =9). Representative immunoblots of 3 blots and quantification of ( d ) liver and ( e ) EWAT pS473 Akt, total Akt and eEF2 at time 180 min during steady state of HIEG clamp ( n =9); relative unit (RU) to that of IL-6Rα f/f (Control). ( f ) Representative liver sections of 22 samples with haematoxylin and eosin (H E) staining; scale bars, 75 μm. ( g ) Hepatic content of triglycerides (TG), free cholesterols (Ch), cholesteryl esters (CE), diglycerides (DAG) and ceramides ( n =6). ( h ) Representative EWAT sections of 20 samples with F4/80 staining and quantification of adipocyte size in area and crown-like structure (CLS) counts ( n =8 versus 12); scale bars, 100 pixels. ( i ) Fast (12 h) and refeed (2 h) plasma TG and cholesterol levels ( n =19 versus 20). Two-tailed t -tests and two-way ANOVA used for statistical analyses (* P
    Figure Legend Snippet: Improved glucose homeostasis and lipid metabolism in IL-6Rα T-KO (8-week HFD). ( a ) Blood glucose (BG) and glucose infusion rate (GIR) during hyperinsulinaemic-euglycaemic (HIEG) clamp experiments ( n =9); two-way analysis of variance (ANOVA) was performed on data from the green-shaded steady state (130–180 min). ( b ) Rate of hepatic glucose production (HGP) at basal and during steady state of HIEG clamp ( n =9). ( c ) Rate of glucose uptake (GU) during steady state of HIEG clamp ( n =9). Representative immunoblots of 3 blots and quantification of ( d ) liver and ( e ) EWAT pS473 Akt, total Akt and eEF2 at time 180 min during steady state of HIEG clamp ( n =9); relative unit (RU) to that of IL-6Rα f/f (Control). ( f ) Representative liver sections of 22 samples with haematoxylin and eosin (H E) staining; scale bars, 75 μm. ( g ) Hepatic content of triglycerides (TG), free cholesterols (Ch), cholesteryl esters (CE), diglycerides (DAG) and ceramides ( n =6). ( h ) Representative EWAT sections of 20 samples with F4/80 staining and quantification of adipocyte size in area and crown-like structure (CLS) counts ( n =8 versus 12); scale bars, 100 pixels. ( i ) Fast (12 h) and refeed (2 h) plasma TG and cholesterol levels ( n =19 versus 20). Two-tailed t -tests and two-way ANOVA used for statistical analyses (* P

    Techniques Used: Western Blot, Staining, Two Tailed Test

    Physiological parameters and metabolic characterization of IL-6Rα T-KO mice (16-week HFD). ( a ) BW curve from 8 to 16 weeks on HFD and percentage of body weight gain ( n =22 versus 25). ( b ) Fasting (6 h) blood glucose ( n =22 versus 25), plasma insulin and HOMA-IR ( n =16 versus 19). ( c ) Blood glucose concentrations during IPGTT after a 6-h fast ( n =22 versus 25). ( d ) Blood glucose as a percentage of basal value during IPITT after a 2-h fast ( n =16 versus 21). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P
    Figure Legend Snippet: Physiological parameters and metabolic characterization of IL-6Rα T-KO mice (16-week HFD). ( a ) BW curve from 8 to 16 weeks on HFD and percentage of body weight gain ( n =22 versus 25). ( b ) Fasting (6 h) blood glucose ( n =22 versus 25), plasma insulin and HOMA-IR ( n =16 versus 19). ( c ) Blood glucose concentrations during IPGTT after a 6-h fast ( n =22 versus 25). ( d ) Blood glucose as a percentage of basal value during IPITT after a 2-h fast ( n =16 versus 21). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P

    Techniques Used: Mouse Assay, Two Tailed Test

    Inflammatory profile of liver and EWAT (16-week HFD). Flow cytometry analyses of ( a ) liver and ( b ) EWAT composition of total CD11c+ myeloid cells, F4/80+ myeloid cells, total MΦ, CD11c+ MΦ and F4/80+ DC, presented as PLICs ( n =3 analysed samples pooled from n =4–8 animals per sample). Flow cytometry analyses of ( c ) liver and ( d ) EWAT composition of total T cells, CD8+ and CD4+ T cells, Th1, Th17, Th2 and CD25+ Treg, presented as PLIC CD45+ ( n =3–5 analysed samples pooled from n =4–8 animals per sample); ND, not detected. JNK activity in ( e ) liver and ( f ) EWAT with the measurement of pS63 c-Jun by immunoblotting (representative immunoblots of 2 blots) and quantification ( n =8); relative unit (RU) to that of IL-6Rα f/f (Control). ( g ) Liver and ( h ) EWAT gene expression profile ( n =11). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P
    Figure Legend Snippet: Inflammatory profile of liver and EWAT (16-week HFD). Flow cytometry analyses of ( a ) liver and ( b ) EWAT composition of total CD11c+ myeloid cells, F4/80+ myeloid cells, total MΦ, CD11c+ MΦ and F4/80+ DC, presented as PLICs ( n =3 analysed samples pooled from n =4–8 animals per sample). Flow cytometry analyses of ( c ) liver and ( d ) EWAT composition of total T cells, CD8+ and CD4+ T cells, Th1, Th17, Th2 and CD25+ Treg, presented as PLIC CD45+ ( n =3–5 analysed samples pooled from n =4–8 animals per sample); ND, not detected. JNK activity in ( e ) liver and ( f ) EWAT with the measurement of pS63 c-Jun by immunoblotting (representative immunoblots of 2 blots) and quantification ( n =8); relative unit (RU) to that of IL-6Rα f/f (Control). ( g ) Liver and ( h ) EWAT gene expression profile ( n =11). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P

    Techniques Used: Flow Cytometry, Cytometry, Activity Assay, Western Blot, Expressing, Two Tailed Test

    Severe glucose homeostasis and lipid metabolism in IL-6Rα T-KO (16-week HFD). ( a ) BG and GIR during HIEG clamp experiments ( n =6 versus 9); two-way analysis of variance (ANOVA) was performed on data from the green-shaded steady state (130–180 min). ( b ) Rate of HGP at basal and during steady state of HIEG clamp ( n =6 versus 9). ( c ) Rate of GU during steady state of HIEG clamp ( n =6 versus 9). Representative immunoblots of 3 blots and quantification of ( d ) liver and ( e ) EWAT pS473 Akt, total Akt and eEF2 at time 180 min during steady state of HIEG clamp ( n =6 versus 9); relative unit (RU) to that of IL-6Rα f/f (Control). ( f ) Representative liver sections of 21 samples with haematoxylin and eosin (H E) staining; scale bars, 75 μm. ( g ) Hepatic content of TG, Ch, CE, DAG and ceramides ( n =9 versus 10). ( h ) Representative EWAT sections of 16 samples with F4/80 staining and quantification of adipocyte size in area and CLS counts ( n =7 versus 9); scale bars, 100 pixels. ( i ) Fast (12 h) and refeed (2 h) plasma TG and cholesterol levels ( n =16 versus 17). Two-tailed t -tests and two-way ANOVA used for statistical analyses (*** P
    Figure Legend Snippet: Severe glucose homeostasis and lipid metabolism in IL-6Rα T-KO (16-week HFD). ( a ) BG and GIR during HIEG clamp experiments ( n =6 versus 9); two-way analysis of variance (ANOVA) was performed on data from the green-shaded steady state (130–180 min). ( b ) Rate of HGP at basal and during steady state of HIEG clamp ( n =6 versus 9). ( c ) Rate of GU during steady state of HIEG clamp ( n =6 versus 9). Representative immunoblots of 3 blots and quantification of ( d ) liver and ( e ) EWAT pS473 Akt, total Akt and eEF2 at time 180 min during steady state of HIEG clamp ( n =6 versus 9); relative unit (RU) to that of IL-6Rα f/f (Control). ( f ) Representative liver sections of 21 samples with haematoxylin and eosin (H E) staining; scale bars, 75 μm. ( g ) Hepatic content of TG, Ch, CE, DAG and ceramides ( n =9 versus 10). ( h ) Representative EWAT sections of 16 samples with F4/80 staining and quantification of adipocyte size in area and CLS counts ( n =7 versus 9); scale bars, 100 pixels. ( i ) Fast (12 h) and refeed (2 h) plasma TG and cholesterol levels ( n =16 versus 17). Two-tailed t -tests and two-way ANOVA used for statistical analyses (*** P

    Techniques Used: Western Blot, Staining, Two Tailed Test

    Physiological parameters and metabolic characterization of IL-6Rα T-KO mice (8-week HFD). ( a ) Body weight (BW) curve on HFD and percentage of body weight gain ( n =21 versus 26). ( b ) Fasting (6 h) blood glucose ( n =21 versus 26), plasma insulin and HOMA-IR ( n =18 versus 16). ( c ) Blood glucose concentrations during intraperitoneal glucose tolerance tests (IPGTT) after a 6 h fast ( n =21 versus 26). ( d ) Blood glucose as a percentage of basal value during intraperitoneal insulin tolerance tests (IPITT) after a 2 h fast ( n =15 versus 18). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P
    Figure Legend Snippet: Physiological parameters and metabolic characterization of IL-6Rα T-KO mice (8-week HFD). ( a ) Body weight (BW) curve on HFD and percentage of body weight gain ( n =21 versus 26). ( b ) Fasting (6 h) blood glucose ( n =21 versus 26), plasma insulin and HOMA-IR ( n =18 versus 16). ( c ) Blood glucose concentrations during intraperitoneal glucose tolerance tests (IPGTT) after a 6 h fast ( n =21 versus 26). ( d ) Blood glucose as a percentage of basal value during intraperitoneal insulin tolerance tests (IPITT) after a 2 h fast ( n =15 versus 18). Two-tailed t -tests and two-way analysis of variance (ANOVA) used for statistical analyses (* P

    Techniques Used: Mouse Assay, Two Tailed Test

    Increased IL-6 and sIL-6Rα levels upon prolonged HFD feeding (8- and 16-week HFD). ( a ) Serum IL-6 levels ( n =11 versus 10–14). ( b ) Serum sIL-6Rα levels ( n =20 versus 20). ( c ) Liver IL-6 content ( n =11–12 versus 11–12). ( d ) EWAT IL-6 content ( n =11–12 versus 11–12). Representative immunoblots and quantification of ( e ) liver ( n =8 versus 8) and ( f ) EWAT sIL-6Rα and eEF2 ( n =4–8 versus 4–8) with a serum sample (S); relative unit (RU) to that of IL-6Rα f/f (Control). Flow cytometry analyses of gp130 expression in cells isolated from ( g ) liver ( n =10–12 versus 10–12) and ( h ) EWAT ( n =10–12 versus 10–13), presented as gp130+ cells in percentage of live CD3+ immune cells (PLIC CD3+) and as amount of gp130 detection in median fluorescence intensity per positive cell (MFI/+Cell). Two-way analysis of variance (ANOVA) with multiple analysis used for statistical analyses (* P
    Figure Legend Snippet: Increased IL-6 and sIL-6Rα levels upon prolonged HFD feeding (8- and 16-week HFD). ( a ) Serum IL-6 levels ( n =11 versus 10–14). ( b ) Serum sIL-6Rα levels ( n =20 versus 20). ( c ) Liver IL-6 content ( n =11–12 versus 11–12). ( d ) EWAT IL-6 content ( n =11–12 versus 11–12). Representative immunoblots and quantification of ( e ) liver ( n =8 versus 8) and ( f ) EWAT sIL-6Rα and eEF2 ( n =4–8 versus 4–8) with a serum sample (S); relative unit (RU) to that of IL-6Rα f/f (Control). Flow cytometry analyses of gp130 expression in cells isolated from ( g ) liver ( n =10–12 versus 10–12) and ( h ) EWAT ( n =10–12 versus 10–13), presented as gp130+ cells in percentage of live CD3+ immune cells (PLIC CD3+) and as amount of gp130 detection in median fluorescence intensity per positive cell (MFI/+Cell). Two-way analysis of variance (ANOVA) with multiple analysis used for statistical analyses (* P

    Techniques Used: Western Blot, Flow Cytometry, Cytometry, Expressing, Isolation, Fluorescence

    12) Product Images from "Regulatory effect of anti-gp130 functional mAb on IL-6 mediated RANKL and Wnt5a expression through JAK-STAT3 signaling pathway in FLS"

    Article Title: Regulatory effect of anti-gp130 functional mAb on IL-6 mediated RANKL and Wnt5a expression through JAK-STAT3 signaling pathway in FLS

    Journal: Oncotarget

    doi: 10.18632/oncotarget.23917

    M10 intervention of the IL-6 signaling pathway in RA FLS (A) Western blotting determination of Bcl-2 levels in RA PBMC and FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 72 h. (B) Western blot detection of STAT3 phosphorylation in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Western blot detection of STAT3 phosphorylation in normal PBMC, RA PBMC, RA SFMC, and RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 30 min. (D) FACS detection of the effect of M10 on STAT3 phosphorylation in normal PBMC, RA PBMC, and RA SFMC.
    Figure Legend Snippet: M10 intervention of the IL-6 signaling pathway in RA FLS (A) Western blotting determination of Bcl-2 levels in RA PBMC and FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 72 h. (B) Western blot detection of STAT3 phosphorylation in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Western blot detection of STAT3 phosphorylation in normal PBMC, RA PBMC, RA SFMC, and RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα (100 ng/mL) for 30 min. (D) FACS detection of the effect of M10 on STAT3 phosphorylation in normal PBMC, RA PBMC, and RA SFMC.

    Techniques Used: Western Blot, Cell Culture, FACS

    Changes in IL-6/sIL-6Rα–induced RANKL and WNT5A expression after M10 pretreatment of RA FLS (A) Western blot detection of WNT5A and RANKL expression in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (B) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS pretreated with M10, and then cultured with IL-6/sIL-6Rα for 72 h. Data are normalized to β-actin and reported in relative expression units. (D) Immunostaining of RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα for 72 h (×200 magnification). Figures are representative of three independent experiments. (E) The number of RANKL- and WNT5A-positive staining cells.
    Figure Legend Snippet: Changes in IL-6/sIL-6Rα–induced RANKL and WNT5A expression after M10 pretreatment of RA FLS (A) Western blot detection of WNT5A and RANKL expression in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (B) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS following 3-day stimulation with IL-6 and sIL-6Rα. (C) Real-time PCR determination of RANKL and WNT5A mRNA in RA FLS pretreated with M10, and then cultured with IL-6/sIL-6Rα for 72 h. Data are normalized to β-actin and reported in relative expression units. (D) Immunostaining of RA FLS pretreated with M10 and cultured with IL-6/sIL-6Rα for 72 h (×200 magnification). Figures are representative of three independent experiments. (E) The number of RANKL- and WNT5A-positive staining cells.

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Immunostaining, Staining

    Schematic representation of the IL-6/sIL-6Rα/gp130 signaling pathways in RA FLS RA FLS release IL-6, which then interacts with IL-6Rα and gp130, forming a hexameric signaling complex and subsequently activating the JAK–STAT pathway and initiating RANKL, WNT5A, and Bcl-2 production.
    Figure Legend Snippet: Schematic representation of the IL-6/sIL-6Rα/gp130 signaling pathways in RA FLS RA FLS release IL-6, which then interacts with IL-6Rα and gp130, forming a hexameric signaling complex and subsequently activating the JAK–STAT pathway and initiating RANKL, WNT5A, and Bcl-2 production.

    Techniques Used:

    Analysis of IL-6/IL-6Rα/gp130 expression levels and RANKL in patients with RA (A) IL-6 levels in RA SF and in the control, OA, and RA serum. (B, C) sIL-6Rα and sgp130 levels in the control, OA, and RA serum and in the OA and RA SF. (D) Analysis of the sIL-6Rα and sgp130 ratio. (E) ELISA determination of soluble RANKL concentrations in RA and OA serum and SF. (F) Detection of MMP3 and TIMP1 in RA or OA serum and SF. (G) Western blot detection of RANKL expression in the bone tissues of patients with RA and the controls. (H) Bcl-2 levels in RA SF and in control, OA, and RA serum. (I) gp130 expression in the cells from the controls and patients with RA after 10-day induction with RANKL and GM-CSF; the percentage of gp130-positive cells was detected.
    Figure Legend Snippet: Analysis of IL-6/IL-6Rα/gp130 expression levels and RANKL in patients with RA (A) IL-6 levels in RA SF and in the control, OA, and RA serum. (B, C) sIL-6Rα and sgp130 levels in the control, OA, and RA serum and in the OA and RA SF. (D) Analysis of the sIL-6Rα and sgp130 ratio. (E) ELISA determination of soluble RANKL concentrations in RA and OA serum and SF. (F) Detection of MMP3 and TIMP1 in RA or OA serum and SF. (G) Western blot detection of RANKL expression in the bone tissues of patients with RA and the controls. (H) Bcl-2 levels in RA SF and in control, OA, and RA serum. (I) gp130 expression in the cells from the controls and patients with RA after 10-day induction with RANKL and GM-CSF; the percentage of gp130-positive cells was detected.

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

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    Article Title: IL-6 Trans-Signaling and Crosstalk Among Tumor, Muscle and Fat Mediate Pancreatic Cancer Cachexia
    Article Snippet: .. Fully differentiated myotubes were then treated in triplicate with either DF media (control), recombinant mouse IL-6 (300 pg/mL) protein (406-ML-005, R & D Systems; Minneapolis, MN USA), recombinant mouse IL6R (25 ng/mL) (P9767-5G, Sigma Aldrich; St. Louis, MO, USA), the combination of IL-6 (300 pg/mL) and IL6R (25 ng/mL) (1830-SR-025, R & D Systems; Minneapolis, MN, USA) or IL-6 (300 pg/mL) in the presence of IL-6 neutralizing antibody (2 mg/mL) (14-7061-85, Thermo Fisher; Waltham, MA, USA) for 48 hours. ..

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    Article Snippet: Recombinant IL-6 treatment Recombinant mouse IL-6 was purchased from R & D Systems (No. 406-ML-025, USA) and dissolved IL-6 in 0.9% NaCl solution. .. In the IL-6 group, 10 μg/kg recombinant mouse IL-6 was intravenously administered 15 min prior to the onset of myocardial ischemia, whereas the control group received the same volume of 0.9% NaCl over the same period as described previously [ ]. .. When the injection was complete, we began the in situ myocardial ischemia induction.

    Flow Cytometry:

    Article Title: Temporal and tissue-specific requirements for T-lymphocyte IL-6 signalling in obesity-associated inflammation and insulin resistance
    Article Snippet: .. Detection of STAT-1/3 phosphorylation by flow cytometry Following the manufacturer's guidelines of BD Phosflow Protocol II, the mild alcohol method (BD Biosciences), 106 enriched splenic T cells in 100 μl per well on a conical-bottomed 96-well plate were cultured for 30 min in serum-free (SF) culture medium (RPMI-1640, phenol red free, 1% glutamine and 5% penicillin–streptomycin) before being transferred to another plate with control SF medium (unstimulated), SF media containing freshly prepared 70 ng ml−1 IL-6 (406-ML-005, R & D Systems), 200 ng ml−1 IL-6—IL-6Rα complex (9038-SR-025, R & D Systems) or 130 ng ml−1 sIL-6Rα alone (1830-SR-025, R & D Systems). .. After stimulation (20 min), cells were pelleted and stained for extracellular markers before lysis, fixation and permeabilization for intracellular staining of pY701 STAT-1 and pY705 STAT-3.

    Cytometry:

    Article Title: Temporal and tissue-specific requirements for T-lymphocyte IL-6 signalling in obesity-associated inflammation and insulin resistance
    Article Snippet: .. Detection of STAT-1/3 phosphorylation by flow cytometry Following the manufacturer's guidelines of BD Phosflow Protocol II, the mild alcohol method (BD Biosciences), 106 enriched splenic T cells in 100 μl per well on a conical-bottomed 96-well plate were cultured for 30 min in serum-free (SF) culture medium (RPMI-1640, phenol red free, 1% glutamine and 5% penicillin–streptomycin) before being transferred to another plate with control SF medium (unstimulated), SF media containing freshly prepared 70 ng ml−1 IL-6 (406-ML-005, R & D Systems), 200 ng ml−1 IL-6—IL-6Rα complex (9038-SR-025, R & D Systems) or 130 ng ml−1 sIL-6Rα alone (1830-SR-025, R & D Systems). .. After stimulation (20 min), cells were pelleted and stained for extracellular markers before lysis, fixation and permeabilization for intracellular staining of pY701 STAT-1 and pY705 STAT-3.

    Cell Culture:

    Article Title: Temporal and tissue-specific requirements for T-lymphocyte IL-6 signalling in obesity-associated inflammation and insulin resistance
    Article Snippet: .. Detection of STAT-1/3 phosphorylation by flow cytometry Following the manufacturer's guidelines of BD Phosflow Protocol II, the mild alcohol method (BD Biosciences), 106 enriched splenic T cells in 100 μl per well on a conical-bottomed 96-well plate were cultured for 30 min in serum-free (SF) culture medium (RPMI-1640, phenol red free, 1% glutamine and 5% penicillin–streptomycin) before being transferred to another plate with control SF medium (unstimulated), SF media containing freshly prepared 70 ng ml−1 IL-6 (406-ML-005, R & D Systems), 200 ng ml−1 IL-6—IL-6Rα complex (9038-SR-025, R & D Systems) or 130 ng ml−1 sIL-6Rα alone (1830-SR-025, R & D Systems). .. After stimulation (20 min), cells were pelleted and stained for extracellular markers before lysis, fixation and permeabilization for intracellular staining of pY701 STAT-1 and pY705 STAT-3.

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    R&D Systems sil 6rα
    IL-6 treatment stimulates bone formation in vivo but requires the addition of sIL-6R. A , schematic diagram. B , calvarial thickness in 6-week-old male C57BL/6 mice with vehicle (saline), hIL-6 alone (0.2 μg/day), hIL-6 (0.2 μg/day) with <t>sIL-6rα</t> (0.1 μg/day), hyper-IL-6 (0.2 μg/day), or murine OSM (0.2 μg/day) by subcutaneous calvarial injections. Data are shown as individual data points for each animal with mean ± S.E. ( error bars ); *, p
    Sil 6rα, supplied by R&D Systems, 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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    R&D Systems soluble il 6rα
    IL-6 signaling is critical for chemotaxis of DCs. A , Higher IL-6 signaling of WT BMDCs than that of fascin1 KO BMDCs. Proximity Ligation Assay (PLA) was used to determine the in situ association between <t>IL-6Rα</t> and gp130, representing the extent of IL-6 signaling. a b, immature BMDCs; c d, mature BMDCs. a c, WT. b d, fascin1 KO. Fluorescence speckles (arrows) indicate the association between IL-6Rα and gp130. Cell boundaries are indicated by dashed lines. B , Quantitative analyses of PLA signals in WT and fascin1 KO BMDCs (n=51). **, p
    Soluble Il 6rα, supplied by R&D Systems, 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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    IL-6 treatment stimulates bone formation in vivo but requires the addition of sIL-6R. A , schematic diagram. B , calvarial thickness in 6-week-old male C57BL/6 mice with vehicle (saline), hIL-6 alone (0.2 μg/day), hIL-6 (0.2 μg/day) with sIL-6rα (0.1 μg/day), hyper-IL-6 (0.2 μg/day), or murine OSM (0.2 μg/day) by subcutaneous calvarial injections. Data are shown as individual data points for each animal with mean ± S.E. ( error bars ); *, p

    Journal: The Journal of Biological Chemistry

    Article Title: IL-6 exhibits both cis- and trans-signaling in osteocytes and osteoblasts, but only trans-signaling promotes bone formation and osteoclastogenesis

    doi: 10.1074/jbc.RA119.008074

    Figure Lengend Snippet: IL-6 treatment stimulates bone formation in vivo but requires the addition of sIL-6R. A , schematic diagram. B , calvarial thickness in 6-week-old male C57BL/6 mice with vehicle (saline), hIL-6 alone (0.2 μg/day), hIL-6 (0.2 μg/day) with sIL-6rα (0.1 μg/day), hyper-IL-6 (0.2 μg/day), or murine OSM (0.2 μg/day) by subcutaneous calvarial injections. Data are shown as individual data points for each animal with mean ± S.E. ( error bars ); *, p

    Article Snippet: In vivo assessment of IL-6 effects on bone formation To determine whether IL-6 cis - or trans- signaling can stimulate bone formation in vivo, 6-week-old male C57/BL6 mice were subjected to subcutaneous calvarial injections for five consecutive days as described previously ( , ) with vehicle (saline), hIL-6 alone (0.2 μg/day, hIL-6, R & D Systems), hIL-6 (0.2 μg/day) with sIL-6rα (0.1 μg/day, R & D Systems), hyper-IL-6 (0.2 μg/day, kindly provided by Dr. Rose-John , or murine OSM as a positive control (0.2 μg/day) ( ).

    Techniques: In Vivo, Mouse Assay

    IL-27p28 and IL-6 sequence homology and proposed model of IL-30 and IL-27 interactions with IL-27 receptor α/T cell cytokine receptor (WSX-1), glycoprotein 130 (gp130), and soluble IL-6 receptor α (sIL-6Rα). (A) Human IL-27p28 (accession number: NP_663634.2) and human IL-6 (accession number: NP_000591.1) sequences were aligned using the T-COFFEE-expresso program ( 32 ). α-helices A, B, C, and D are indicated by bold red letters and boxed sequences. Conserved Trp 197 on IL-27p28 and Trp 185 on IL-6 are indicated by the red arrow. [ (B) ; left] The p28 subunit contains three binding sites (site 1, site 2, and site 3). For the binding of the dimeric IL-27, it has been demonstrated that the p28 site 1 interacts with Epstein–Barr virus-induced gene 3 (EBI3), site 2 interacts with WSX-1, and site 3 interacts with gp130 ( 36 ). For the binding of IL-30 (IL-27p28) to the WSX-1 and gp130 receptor chains, sIL-6Rα may be required. Since sIL-6Rα interacts with site 1 on IL-6, and IL-6, and IL-30 share homology, the p28 site 2 may interact with sIL-6Rα to enable IL-30-mediated signaling. Furthermore, sIL-6Rα may serve as an alternative binding partner in the absence of EBI3 to induce IL-30 signaling. [ (B) ; right] IL-6 may compete with IL-30 for interaction with sIL-6Rα due to higher affinity for IL-6. As a result, in the presence of IL-6, both IL-30 and IL-27 signaling and downstream functions are inhibited.

    Journal: Frontiers in Immunology

    Article Title: Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes

    doi: 10.3389/fimmu.2018.00256

    Figure Lengend Snippet: IL-27p28 and IL-6 sequence homology and proposed model of IL-30 and IL-27 interactions with IL-27 receptor α/T cell cytokine receptor (WSX-1), glycoprotein 130 (gp130), and soluble IL-6 receptor α (sIL-6Rα). (A) Human IL-27p28 (accession number: NP_663634.2) and human IL-6 (accession number: NP_000591.1) sequences were aligned using the T-COFFEE-expresso program ( 32 ). α-helices A, B, C, and D are indicated by bold red letters and boxed sequences. Conserved Trp 197 on IL-27p28 and Trp 185 on IL-6 are indicated by the red arrow. [ (B) ; left] The p28 subunit contains three binding sites (site 1, site 2, and site 3). For the binding of the dimeric IL-27, it has been demonstrated that the p28 site 1 interacts with Epstein–Barr virus-induced gene 3 (EBI3), site 2 interacts with WSX-1, and site 3 interacts with gp130 ( 36 ). For the binding of IL-30 (IL-27p28) to the WSX-1 and gp130 receptor chains, sIL-6Rα may be required. Since sIL-6Rα interacts with site 1 on IL-6, and IL-6, and IL-30 share homology, the p28 site 2 may interact with sIL-6Rα to enable IL-30-mediated signaling. Furthermore, sIL-6Rα may serve as an alternative binding partner in the absence of EBI3 to induce IL-30 signaling. [ (B) ; right] IL-6 may compete with IL-30 for interaction with sIL-6Rα due to higher affinity for IL-6. As a result, in the presence of IL-6, both IL-30 and IL-27 signaling and downstream functions are inhibited.

    Article Snippet: ELISA Culture supernatants were used to quantify cytokine expression according to manufacturers’ instructions for human TNF-α (Thermo Fisher Scientific, Affymetrix eBioscience, Waltham, MA, USA; Ready-Set-Go kit; sensitivity: 4–500 pg/mL), human CXCL10/IP-10 (R & D Systems, DuoSet kit; sensitivity: 31.2–2,000 pg/mL), and sIL-6Rα (R & D systems, DuoSet kit; sensitivity: 15.6–1,000 pg/mL).

    Techniques: Sequencing, Binding Assay

    Recombinant IL-6 inhibits IL-27- and IL-30-IP-10 production. (A) THP-1 cells and (B) primary human monocytes were pretreated with recombinant IL-6 (10 ng/mL) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of 13 different THP-1 experiments or 6 different monocyte donors. THP-1 cells and primary human monocytes were stimulated with or without recombinant IL-6 (10 ng/mL) for 30 min and then stained with IL-27 receptor α/T cell cytokine receptor (WSX-1) (C) , glycoprotein 130 (gp130) (D) , or anti-IL-6Rα (E) . Mann–Whitney U tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated THP-1 cells. Wilcoxon signed-rank matched pair tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated primary monocytes. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Journal: Frontiers in Immunology

    Article Title: Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes

    doi: 10.3389/fimmu.2018.00256

    Figure Lengend Snippet: Recombinant IL-6 inhibits IL-27- and IL-30-IP-10 production. (A) THP-1 cells and (B) primary human monocytes were pretreated with recombinant IL-6 (10 ng/mL) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of 13 different THP-1 experiments or 6 different monocyte donors. THP-1 cells and primary human monocytes were stimulated with or without recombinant IL-6 (10 ng/mL) for 30 min and then stained with IL-27 receptor α/T cell cytokine receptor (WSX-1) (C) , glycoprotein 130 (gp130) (D) , or anti-IL-6Rα (E) . Mann–Whitney U tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated THP-1 cells. Wilcoxon signed-rank matched pair tests were used for statistical analyses between corresponding medium controls and IL-6 pretreated primary monocytes. ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Article Snippet: ELISA Culture supernatants were used to quantify cytokine expression according to manufacturers’ instructions for human TNF-α (Thermo Fisher Scientific, Affymetrix eBioscience, Waltham, MA, USA; Ready-Set-Go kit; sensitivity: 4–500 pg/mL), human CXCL10/IP-10 (R & D Systems, DuoSet kit; sensitivity: 31.2–2,000 pg/mL), and sIL-6Rα (R & D systems, DuoSet kit; sensitivity: 15.6–1,000 pg/mL).

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

    Cleaved soluble IL-6 receptor α (sIL-6Rα) is involved IL-27 and IL-30 function. (A) THP-1 cells were cultured in medium (RPMI + 10% FBS) for 4, 8, 16, and 24 h. Endogenously produced sIL-6Rα was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of three different THP-1 experiments. (B) THP-1 cells and (C) primary human monocytes were treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h, and sIL-6Rα was measured in cell-free supernatants by ELISA. (D,E) Primary human monocytes were pretreated with BB-94 (25 µM) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. (D) sIL-6Rα production and (E) IP-10 production were measured in cell-free supernatants by ELISA. (F,G) Recombinant human IL-6Rα was preincubated with IL-27 or IL-30 for 2 h. THP-1 cells were pretreated with BB-94 (25 µM) for 1 h and then stimulated with preincubated IL-6Rα (2.5 ng/mL) with or without IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. Pretreatment with DMSO was used as a vehicle control for BB-94. (F) sIL-6Rα production, and (G) IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of at least three different THP-1 experiments or three different monocyte donors. Mann–Whitney U tests were used for statistical analyses between indicated groups (B,C) , between corresponding controls (DMSO) and BB-94 treatment (D,E) , or between pairs as indicated (F,G) , unless otherwise specified. Wilcoxon signed-rank matched pair tests were used for statistical analyses between medium or IL-30 control (DMSO) and BB-94 treatment (E) , between medium, IL-27, or IL-30 DMSO and DMSO + rhIL-6Rα treatment (F) , and between IL-27 or IL-30 BB-94 and BB-94 + rhIL-6Rα treatment (G) . ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Journal: Frontiers in Immunology

    Article Title: Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes

    doi: 10.3389/fimmu.2018.00256

    Figure Lengend Snippet: Cleaved soluble IL-6 receptor α (sIL-6Rα) is involved IL-27 and IL-30 function. (A) THP-1 cells were cultured in medium (RPMI + 10% FBS) for 4, 8, 16, and 24 h. Endogenously produced sIL-6Rα was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of three different THP-1 experiments. (B) THP-1 cells and (C) primary human monocytes were treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h, and sIL-6Rα was measured in cell-free supernatants by ELISA. (D,E) Primary human monocytes were pretreated with BB-94 (25 µM) for 30 min and then treated with or without recombinant IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. (D) sIL-6Rα production and (E) IP-10 production were measured in cell-free supernatants by ELISA. (F,G) Recombinant human IL-6Rα was preincubated with IL-27 or IL-30 for 2 h. THP-1 cells were pretreated with BB-94 (25 µM) for 1 h and then stimulated with preincubated IL-6Rα (2.5 ng/mL) with or without IL-27 (50 ng/mL) or IL-30 (50 ng/mL) for 24 h. Pretreatment with DMSO was used as a vehicle control for BB-94. (F) sIL-6Rα production, and (G) IP-10 production was measured in cell-free supernatants by ELISA. Data presented are the mean ± SEM of at least three different THP-1 experiments or three different monocyte donors. Mann–Whitney U tests were used for statistical analyses between indicated groups (B,C) , between corresponding controls (DMSO) and BB-94 treatment (D,E) , or between pairs as indicated (F,G) , unless otherwise specified. Wilcoxon signed-rank matched pair tests were used for statistical analyses between medium or IL-30 control (DMSO) and BB-94 treatment (E) , between medium, IL-27, or IL-30 DMSO and DMSO + rhIL-6Rα treatment (F) , and between IL-27 or IL-30 BB-94 and BB-94 + rhIL-6Rα treatment (G) . ns, not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

    Article Snippet: ELISA Culture supernatants were used to quantify cytokine expression according to manufacturers’ instructions for human TNF-α (Thermo Fisher Scientific, Affymetrix eBioscience, Waltham, MA, USA; Ready-Set-Go kit; sensitivity: 4–500 pg/mL), human CXCL10/IP-10 (R & D Systems, DuoSet kit; sensitivity: 31.2–2,000 pg/mL), and sIL-6Rα (R & D systems, DuoSet kit; sensitivity: 15.6–1,000 pg/mL).

    Techniques: Cell Culture, Produced, Enzyme-linked Immunosorbent Assay, Recombinant, MANN-WHITNEY

    Production of pro-fibrotic molecules by human neutrophils in response to proinflammatory cytokines. Expression of IL6 , TNF , and TGFB1 was determined in human neutrophils stimulated with TNF-α, IL-6, or TGF-β1 using qRT-PCR ( a ). Human mesothelial cells (MeT5A cells) were incubated with TNF-α, IL-6 plus soluble IL-6Rα (sIL-6Rα), TGF-β1, or IFN-γ, followed by measurement of IL6 ( b ), TNF ( b ), CXCL2 ( b ), TGFB1 ( b ), or COL1A1 expression by qRT-PCR ( c ). Three independent experiments were performed. Data are shown as mean ± SEM. * p

    Journal: Scientific Reports

    Article Title: Anti-interleukin-6 receptor antibody treatment ameliorates postoperative adhesion formation

    doi: 10.1038/s41598-019-54175-1

    Figure Lengend Snippet: Production of pro-fibrotic molecules by human neutrophils in response to proinflammatory cytokines. Expression of IL6 , TNF , and TGFB1 was determined in human neutrophils stimulated with TNF-α, IL-6, or TGF-β1 using qRT-PCR ( a ). Human mesothelial cells (MeT5A cells) were incubated with TNF-α, IL-6 plus soluble IL-6Rα (sIL-6Rα), TGF-β1, or IFN-γ, followed by measurement of IL6 ( b ), TNF ( b ), CXCL2 ( b ), TGFB1 ( b ), or COL1A1 expression by qRT-PCR ( c ). Three independent experiments were performed. Data are shown as mean ± SEM. * p

    Article Snippet: Enzyme-linked immunosorbent assay We measured the abundance of IL-6, TNF-α, TGF-β1 and sIL-6Rα in sera of mice with mouse ELISA kits of IL-6 (M6000B, R & D Systems), TNF-α (MTA00B, R & D Systems), TGF-β1 (MB100B, R & D Systems), and sIL-6Rα (DY1830, R & D Systems), respectively.

    Techniques: Expressing, Quantitative RT-PCR, Incubation

    Anti-IL-6Rα antibody protection against postoperative adhesion formation. Wild-type mice were treated with various doses of anti-IL-6Rα antibody or isotype-matched antibody and subjected to cecum cauterization. ( a ) Adhesion scores were determined on day 7 following the procedure. Representative photos are shown. ( b,c ) Cecum lesions were sampled at the indicated time points for indicated protein staining ( b ) and for qRT-PCR analysis of indicated proinflammatory and pro-fibrotic mRNA transcripts ( c ). Each experimental group contained 3–5 mice, with two independent experiments performed. Representative photos are shown. Data at 0 day or hour postoperation indicated those in untreated control mice. Data are shown as mean ± SEM. * p

    Journal: Scientific Reports

    Article Title: Anti-interleukin-6 receptor antibody treatment ameliorates postoperative adhesion formation

    doi: 10.1038/s41598-019-54175-1

    Figure Lengend Snippet: Anti-IL-6Rα antibody protection against postoperative adhesion formation. Wild-type mice were treated with various doses of anti-IL-6Rα antibody or isotype-matched antibody and subjected to cecum cauterization. ( a ) Adhesion scores were determined on day 7 following the procedure. Representative photos are shown. ( b,c ) Cecum lesions were sampled at the indicated time points for indicated protein staining ( b ) and for qRT-PCR analysis of indicated proinflammatory and pro-fibrotic mRNA transcripts ( c ). Each experimental group contained 3–5 mice, with two independent experiments performed. Representative photos are shown. Data at 0 day or hour postoperation indicated those in untreated control mice. Data are shown as mean ± SEM. * p

    Article Snippet: Enzyme-linked immunosorbent assay We measured the abundance of IL-6, TNF-α, TGF-β1 and sIL-6Rα in sera of mice with mouse ELISA kits of IL-6 (M6000B, R & D Systems), TNF-α (MTA00B, R & D Systems), TGF-β1 (MB100B, R & D Systems), and sIL-6Rα (DY1830, R & D Systems), respectively.

    Techniques: Mouse Assay, Staining, Quantitative RT-PCR

    IL-6 signaling is critical for chemotaxis of DCs. A , Higher IL-6 signaling of WT BMDCs than that of fascin1 KO BMDCs. Proximity Ligation Assay (PLA) was used to determine the in situ association between IL-6Rα and gp130, representing the extent of IL-6 signaling. a b, immature BMDCs; c d, mature BMDCs. a c, WT. b d, fascin1 KO. Fluorescence speckles (arrows) indicate the association between IL-6Rα and gp130. Cell boundaries are indicated by dashed lines. B , Quantitative analyses of PLA signals in WT and fascin1 KO BMDCs (n=51). **, p

    Journal: bioRxiv

    Article Title: Investigation of fascin1, a marker of mature dendritic cells, reveals a New role for IL-6 signaling in chemotaxis

    doi: 10.1101/2020.03.19.979104

    Figure Lengend Snippet: IL-6 signaling is critical for chemotaxis of DCs. A , Higher IL-6 signaling of WT BMDCs than that of fascin1 KO BMDCs. Proximity Ligation Assay (PLA) was used to determine the in situ association between IL-6Rα and gp130, representing the extent of IL-6 signaling. a b, immature BMDCs; c d, mature BMDCs. a c, WT. b d, fascin1 KO. Fluorescence speckles (arrows) indicate the association between IL-6Rα and gp130. Cell boundaries are indicated by dashed lines. B , Quantitative analyses of PLA signals in WT and fascin1 KO BMDCs (n=51). **, p

    Article Snippet: To restore IL-6 signaling of IL-6Rα KO DCs, human IL-6 (15ng/ml, 206-IL, R & D Systems) and soluble IL-6Rα (sIL-6Rα, 25ng/ml, 227-SR, R & D Systems) were added 1hr after LPS addition.

    Techniques: Chemotaxis Assay, Proximity Ligation Assay, In Situ, Fluorescence

    IL-6Rα signaling is critical for CCR7 internalization and CCL19-induced ERK1/2 phosphorylation. A , Effects of the blockage of IL-6 signaling on CCR7 internalization. WT BMDCs were stimulated with CCL19 for 5 min in the presence of either control (a) or neutralizing antibody (b), and then surface CCR7 expression was determined by flow cytometry. Surface expression of CCR7 was examined with CD86 + BMDCs before (-CCL19) and after CCL19 addition (+CCL19). Note that CCR7 internalization was blocked in the absence of a neutralizing antibody to IL-6Rα. B , Effects of IL-6 signaling on CCR7 internalization of IL-6Rα KO BMDCs. IL-6Rα KO BMDCs were stimulated with CCL19 in the absence (a) or presence of sIL-6Rα (soluble IL-6Rα) and IL-6 (b). Note that IL-6Rα KO BMDCs showed impaired CCR7 internalization (a), which was rescued by the addition of sIL-6Rα and IL-6 (b). C , Inhibition of CCR7 internalization by the blockage of IL-6 signaling in HEK293T epithelial cells. HEK293T cells stably expressing mouse CCR7-GFP were stimulated with CCL19 in the presence of either control or neutralizing antibody against IL-6Rα. a-d, fluorescence images of CCR7-GFP in control cells (a b) or IL-6Rα neutralizing antibody-treated cells (c d) before (a c) or after addition of CCL19 (b d). The same cells were counter stained with an antibody specific to mouse CCR7 to detect only surface CCR7 (e-h) in control (e f) or IL-6Rα neutralizing antibody-treated cells (g h) before (e g) or after (f h) addition of CCL19. D , Quantitative measurements of surface CCR7 immunofluorescence (panels e-h of C) at cell-to-cell contacts. In contrast to the control, the addition of a neutralizing antibody against IL-6Rα blocked internalization of CCR7. E , Effects of the blockage of IL-6 signaling on CCL19-induced ERK1/2 phosphorylation of WT BMDCs. WT BMDCs were stimulated with CCL19 in the presence of a control or neutralizing antibody, then ERK1/2 phosphorylation levels were determined at 0, 5, 15 and 30min after CCL19 addition using Western blotting with a phosphospecific ERK1/2 antibody. For normalization, the same membranes were reblotted with a pan ERK1/2 antibody. The ratios of phosphoERK1/2 to total ERK1/2 are indicated below the figure.

    Journal: bioRxiv

    Article Title: Investigation of fascin1, a marker of mature dendritic cells, reveals a New role for IL-6 signaling in chemotaxis

    doi: 10.1101/2020.03.19.979104

    Figure Lengend Snippet: IL-6Rα signaling is critical for CCR7 internalization and CCL19-induced ERK1/2 phosphorylation. A , Effects of the blockage of IL-6 signaling on CCR7 internalization. WT BMDCs were stimulated with CCL19 for 5 min in the presence of either control (a) or neutralizing antibody (b), and then surface CCR7 expression was determined by flow cytometry. Surface expression of CCR7 was examined with CD86 + BMDCs before (-CCL19) and after CCL19 addition (+CCL19). Note that CCR7 internalization was blocked in the absence of a neutralizing antibody to IL-6Rα. B , Effects of IL-6 signaling on CCR7 internalization of IL-6Rα KO BMDCs. IL-6Rα KO BMDCs were stimulated with CCL19 in the absence (a) or presence of sIL-6Rα (soluble IL-6Rα) and IL-6 (b). Note that IL-6Rα KO BMDCs showed impaired CCR7 internalization (a), which was rescued by the addition of sIL-6Rα and IL-6 (b). C , Inhibition of CCR7 internalization by the blockage of IL-6 signaling in HEK293T epithelial cells. HEK293T cells stably expressing mouse CCR7-GFP were stimulated with CCL19 in the presence of either control or neutralizing antibody against IL-6Rα. a-d, fluorescence images of CCR7-GFP in control cells (a b) or IL-6Rα neutralizing antibody-treated cells (c d) before (a c) or after addition of CCL19 (b d). The same cells were counter stained with an antibody specific to mouse CCR7 to detect only surface CCR7 (e-h) in control (e f) or IL-6Rα neutralizing antibody-treated cells (g h) before (e g) or after (f h) addition of CCL19. D , Quantitative measurements of surface CCR7 immunofluorescence (panels e-h of C) at cell-to-cell contacts. In contrast to the control, the addition of a neutralizing antibody against IL-6Rα blocked internalization of CCR7. E , Effects of the blockage of IL-6 signaling on CCL19-induced ERK1/2 phosphorylation of WT BMDCs. WT BMDCs were stimulated with CCL19 in the presence of a control or neutralizing antibody, then ERK1/2 phosphorylation levels were determined at 0, 5, 15 and 30min after CCL19 addition using Western blotting with a phosphospecific ERK1/2 antibody. For normalization, the same membranes were reblotted with a pan ERK1/2 antibody. The ratios of phosphoERK1/2 to total ERK1/2 are indicated below the figure.

    Article Snippet: To restore IL-6 signaling of IL-6Rα KO DCs, human IL-6 (15ng/ml, 206-IL, R & D Systems) and soluble IL-6Rα (sIL-6Rα, 25ng/ml, 227-SR, R & D Systems) were added 1hr after LPS addition.

    Techniques: Expressing, Flow Cytometry, Inhibition, Stable Transfection, Fluorescence, Staining, Immunofluorescence, Western Blot

    IL-6 signaling is required for directional migration of BMDCs. A, Analyses of migration tracks. Live cell imaging of BMDCs migrating in a collagen gel toward CCL19 was performed to obtain migration tracks (a, c e) and rose diagram plots for directionality(b, d f). a b, WT (n=19): c d, fascin1 KO (n=23): e f, WT BMDCs in the presence of a neutralizing antibody against IL-6Rα (n=28). WT BMDCs (a b) showed more consistent migration toward CCL19 than fascin1 KO counterparts (c d). Blockage of IL-6 signaling inhibits directed migration of WT BMDCs (e f). Arrows, the direction of a CCL19 gradient. B-E, Box plot analyses of parallel (B, FMI∥) and perpendicular (C, FMI⊥) forward migration indexes, directness (D), and migration speeds (E). *, p

    Journal: bioRxiv

    Article Title: Investigation of fascin1, a marker of mature dendritic cells, reveals a New role for IL-6 signaling in chemotaxis

    doi: 10.1101/2020.03.19.979104

    Figure Lengend Snippet: IL-6 signaling is required for directional migration of BMDCs. A, Analyses of migration tracks. Live cell imaging of BMDCs migrating in a collagen gel toward CCL19 was performed to obtain migration tracks (a, c e) and rose diagram plots for directionality(b, d f). a b, WT (n=19): c d, fascin1 KO (n=23): e f, WT BMDCs in the presence of a neutralizing antibody against IL-6Rα (n=28). WT BMDCs (a b) showed more consistent migration toward CCL19 than fascin1 KO counterparts (c d). Blockage of IL-6 signaling inhibits directed migration of WT BMDCs (e f). Arrows, the direction of a CCL19 gradient. B-E, Box plot analyses of parallel (B, FMI∥) and perpendicular (C, FMI⊥) forward migration indexes, directness (D), and migration speeds (E). *, p

    Article Snippet: To restore IL-6 signaling of IL-6Rα KO DCs, human IL-6 (15ng/ml, 206-IL, R & D Systems) and soluble IL-6Rα (sIL-6Rα, 25ng/ml, 227-SR, R & D Systems) were added 1hr after LPS addition.

    Techniques: Migration, Live Cell Imaging

    Impaired chemotaxis of IL-6Rα KO BMDCs and its restoration by the addition of soluble IL-6Rα (sIL-6Rα) and IL-6. BMDCs were isolated from heterozygous (hetero) and IL-6Rα KO mice. A , Immunofluorescence of IL-6Rα in heterozygous (a) and IL-6Rα KO BMDCs (b) confirming the lack of IL-6Rα in KO BMDCs. B , Collagen-coated, modified Boyden chamber chemotaxis assays of heterozygous (hetero), IL-6Rα KO, and IL-6Rα KO in the presence of sIL-6Rα and IL-6 (KO+sIL-6R/IL-6). Note that KO BMDCs show reduced chemotaxis, which was rescued by the addition of soluble IL-6Rα (sIL-6Rα) and IL-6. C D , IL-6Rα KO did not alter surface expression of CCR7. Immunofluorescence (C) showing similar levels of surface CCR7 in heterozygous (a) and IL-6Rα KO BMDCs (b). Flow cytometry analyses (D) confirming that IL-6Rα KO did not affect the levels of CCR7 surface expression. After CD86-positive heterozygous (a) and IL-6Rα KO (b) BMDCs were gated, the levels of CCR7 surface expression were examined in histogram (c).

    Journal: bioRxiv

    Article Title: Investigation of fascin1, a marker of mature dendritic cells, reveals a New role for IL-6 signaling in chemotaxis

    doi: 10.1101/2020.03.19.979104

    Figure Lengend Snippet: Impaired chemotaxis of IL-6Rα KO BMDCs and its restoration by the addition of soluble IL-6Rα (sIL-6Rα) and IL-6. BMDCs were isolated from heterozygous (hetero) and IL-6Rα KO mice. A , Immunofluorescence of IL-6Rα in heterozygous (a) and IL-6Rα KO BMDCs (b) confirming the lack of IL-6Rα in KO BMDCs. B , Collagen-coated, modified Boyden chamber chemotaxis assays of heterozygous (hetero), IL-6Rα KO, and IL-6Rα KO in the presence of sIL-6Rα and IL-6 (KO+sIL-6R/IL-6). Note that KO BMDCs show reduced chemotaxis, which was rescued by the addition of soluble IL-6Rα (sIL-6Rα) and IL-6. C D , IL-6Rα KO did not alter surface expression of CCR7. Immunofluorescence (C) showing similar levels of surface CCR7 in heterozygous (a) and IL-6Rα KO BMDCs (b). Flow cytometry analyses (D) confirming that IL-6Rα KO did not affect the levels of CCR7 surface expression. After CD86-positive heterozygous (a) and IL-6Rα KO (b) BMDCs were gated, the levels of CCR7 surface expression were examined in histogram (c).

    Article Snippet: To restore IL-6 signaling of IL-6Rα KO DCs, human IL-6 (15ng/ml, 206-IL, R & D Systems) and soluble IL-6Rα (sIL-6Rα, 25ng/ml, 227-SR, R & D Systems) were added 1hr after LPS addition.

    Techniques: Chemotaxis Assay, Isolation, Mouse Assay, Immunofluorescence, Modification, Expressing, Flow Cytometry