recombinant human sil 6r  (R&D Systems)

 
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    Name:
    Recombinant Human DLL1 Protein CF
    Description:
    The Recombinant Human DLL1 Protein from R D Systems is derived from NS0 The Recombinant Human DLL1 Protein has been validated for the following applications Bioactivity
    Catalog Number:
    1818-DL-050
    Price:
    329
    Category:
    Proteins and Enzymes
    Source:
    NS0-derived Recombinant Human DLL1 Protein
    Applications:
    Bioactivity
    Purity:
    >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie« Blue Staining.
    Conjugate:
    Unconjugated
    Size:
    50 ug
    Buy from Supplier


    Structured Review

    R&D Systems recombinant human sil 6r
    TNF-α interacts with microglia to recruit soluble IL-6 receptor. A , Release of IL-6 soluble receptor (sIL6-R) into the supernatant of BV2 mouse microglia cells within 2 h of stimulation ( n = 6). Basal release (control, 26.0 ± 3.9 pg/ml, mean ± SEM) decreased insignificantly in the presence of minocycline. TNF-α increased <t>sIL-6R</t> release significantly, an effect inhibited by minocycline. There was no additional release of sIL-6R by IL-6. x indicates a significant difference between the indicated groups ( p
    The Recombinant Human DLL1 Protein from R D Systems is derived from NS0 The Recombinant Human DLL1 Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/recombinant human sil 6r/product/R&D Systems
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant human sil 6r - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha"

    Article Title: Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4159-15.2016

    TNF-α interacts with microglia to recruit soluble IL-6 receptor. A , Release of IL-6 soluble receptor (sIL6-R) into the supernatant of BV2 mouse microglia cells within 2 h of stimulation ( n = 6). Basal release (control, 26.0 ± 3.9 pg/ml, mean ± SEM) decreased insignificantly in the presence of minocycline. TNF-α increased sIL-6R release significantly, an effect inhibited by minocycline. There was no additional release of sIL-6R by IL-6. x indicates a significant difference between the indicated groups ( p
    Figure Legend Snippet: TNF-α interacts with microglia to recruit soluble IL-6 receptor. A , Release of IL-6 soluble receptor (sIL6-R) into the supernatant of BV2 mouse microglia cells within 2 h of stimulation ( n = 6). Basal release (control, 26.0 ± 3.9 pg/ml, mean ± SEM) decreased insignificantly in the presence of minocycline. TNF-α increased sIL-6R release significantly, an effect inhibited by minocycline. There was no additional release of sIL-6R by IL-6. x indicates a significant difference between the indicated groups ( p

    Techniques Used:

    Effect of minocycline on TNF-α-induced hyperexcitability. A , There were no changes of neuronal responses to mechanical stimulation after application of minocycline only and no increase after subsequent application of minocycline plus TNF-α ( n = 11), but there was an increase of the responses after TNF-α plus sIL-6R in the presence of minocycline ( n = 7). Data are shown as the mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the minocycline/TNF-α group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the minocycline/TNF-α/sIL-6R group: innocuous pressure knee 42.3 ± 28.2, noxious pressure knee 326.1 ± 98.7, innocuous pressure ankle 112.3 ± 60.3, noxious pressure ankle 554.9 ± 160.5. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p
    Figure Legend Snippet: Effect of minocycline on TNF-α-induced hyperexcitability. A , There were no changes of neuronal responses to mechanical stimulation after application of minocycline only and no increase after subsequent application of minocycline plus TNF-α ( n = 11), but there was an increase of the responses after TNF-α plus sIL-6R in the presence of minocycline ( n = 7). Data are shown as the mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the minocycline/TNF-α group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the minocycline/TNF-α/sIL-6R group: innocuous pressure knee 42.3 ± 28.2, noxious pressure knee 326.1 ± 98.7, innocuous pressure ankle 112.3 ± 60.3, noxious pressure ankle 554.9 ± 160.5. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p

    Techniques Used:

    Increase of spinal IL-6 release during peripheral stimulation in the presence of spinally applied TNF-α and neuronal effects of IL-6 and IL-6/sIL-6R. A , Spinal IL-6 release (means ± SEM) into the spinal cord supernatant in the course of the stimulation protocol and spinal TNF-α application. Samples were extracted after 1 h of baseline (control) and 1 and 2 h after TNF-α application ( n = 10). *Significant increase after TNF-α compared with control before TNF-α ( p
    Figure Legend Snippet: Increase of spinal IL-6 release during peripheral stimulation in the presence of spinally applied TNF-α and neuronal effects of IL-6 and IL-6/sIL-6R. A , Spinal IL-6 release (means ± SEM) into the spinal cord supernatant in the course of the stimulation protocol and spinal TNF-α application. Samples were extracted after 1 h of baseline (control) and 1 and 2 h after TNF-α application ( n = 10). *Significant increase after TNF-α compared with control before TNF-α ( p

    Techniques Used:

    Model of the cooperation of TNF-α and IL-6 in the generation of spinal hyperexcitability. TNF-α leads to IL-6 release mainly from neurons and acts on microglia to provide sIL-6R for IL-6 trans -signaling by ADAM10/17-protease-dependent cleavage of IL-6R, which is not present in neurons. Interaction of IL-6-bound IL-6R or IL-6/sIL-6R complex with ubiquitously expressed and homodimerized gp130 is required for IL-6 signaling.
    Figure Legend Snippet: Model of the cooperation of TNF-α and IL-6 in the generation of spinal hyperexcitability. TNF-α leads to IL-6 release mainly from neurons and acts on microglia to provide sIL-6R for IL-6 trans -signaling by ADAM10/17-protease-dependent cleavage of IL-6R, which is not present in neurons. Interaction of IL-6-bound IL-6R or IL-6/sIL-6R complex with ubiquitously expressed and homodimerized gp130 is required for IL-6 signaling.

    Techniques Used:

    Induction of hyperexcitability of deep dorsal horn neurons by TNF-α via IL-6/sIL-6R. A , Hypothetical flow chart of an upstream function of TNF-α initiating the release of IL-6 and sIL-6R from different sources to form the downstream IL-6/sIL-6R complex for IL-6 trans -signaling. Indicated are the action sites of specific inhibitors. B , Increases of neuronal responses to mechanical stimulation by spinal application of IL-6/sIL-6R ( n = 6) and spinal application of IL-6/sIL-6R plus etanercept ( n = 6) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the IL-6/sIL-6R group: innocuous pressure knee 154.5 ± 23.2, noxious pressure knee 357.8 ± 43.1, innocuous pressure ankle 67.0 ± 28.7, noxious pressure ankle 470.4 ± 80.7. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus etanercept group: innocuous pressure knee 81.6 ± 47.8, noxious pressure knee 298.3 ± 84.6, innocuous pressure ankle 53.8 ± 44.0, noxious pressure ankle 322.7 ± 135.4. C , Increases of neuronal responses to mechanical stimulation by spinal application of TNF-α plus minocycline ( n = 11) or IL-6/sIL-6R plus minocycline ( n = 8) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the TNF-α plus minocycline group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus minocycline group: innocuous pressure knee 98.4 ± 34.4, noxious pressure knee 292.5 ± 53.1, innocuous pressure ankle 56.4 ± 36.1, noxious pressure ankle 261.2 ± 54.4. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p
    Figure Legend Snippet: Induction of hyperexcitability of deep dorsal horn neurons by TNF-α via IL-6/sIL-6R. A , Hypothetical flow chart of an upstream function of TNF-α initiating the release of IL-6 and sIL-6R from different sources to form the downstream IL-6/sIL-6R complex for IL-6 trans -signaling. Indicated are the action sites of specific inhibitors. B , Increases of neuronal responses to mechanical stimulation by spinal application of IL-6/sIL-6R ( n = 6) and spinal application of IL-6/sIL-6R plus etanercept ( n = 6) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the IL-6/sIL-6R group: innocuous pressure knee 154.5 ± 23.2, noxious pressure knee 357.8 ± 43.1, innocuous pressure ankle 67.0 ± 28.7, noxious pressure ankle 470.4 ± 80.7. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus etanercept group: innocuous pressure knee 81.6 ± 47.8, noxious pressure knee 298.3 ± 84.6, innocuous pressure ankle 53.8 ± 44.0, noxious pressure ankle 322.7 ± 135.4. C , Increases of neuronal responses to mechanical stimulation by spinal application of TNF-α plus minocycline ( n = 11) or IL-6/sIL-6R plus minocycline ( n = 8) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the TNF-α plus minocycline group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus minocycline group: innocuous pressure knee 98.4 ± 34.4, noxious pressure knee 292.5 ± 53.1, innocuous pressure ankle 56.4 ± 36.1, noxious pressure ankle 261.2 ± 54.4. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p

    Techniques Used: Flow Cytometry

    2) Product Images from "SorLA in Interleukin-6 Signaling and Turnover"

    Article Title: SorLA in Interleukin-6 Signaling and Turnover

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00641-16

    SPR analysis of the binding of sIL-6R to sSorLA and to the Vps10p-D of SorLA (A to D) and SorLA-mediated uptake of sIL-6R in cells (E). The concentration dependence of sIL-6R binding to immobilized sSorLA (A) and the SorLA Vps10p-D (C) was evaluated. The indicated K d values were calculated on the basis of the collected sums of data. The inhibition by SorLA propeptide (pro) of sIL-6R binding to sSorLA (B) and to the Vps10p-D (D) was also evaluated. SorLA was preincubated with a saturating concentration of propeptide (2 μM) prior to the injection of a mixture of propeptide (2 μM) and sIL-6R (100 nM). The response obtained with sIL-6R alone, i.e., the curve to be expected in the absence of inhibition, is indicated in each case in red. (E) Uptake of sIL-6R in SorLA-transfected and wt HEK293 cells. The cells were incubated (37°C, 30 min) at 250 nM sIL-6R in the absence or presence of 20 μM SorLA propeptide. The cells were then washed, fixed, permeabilized, and subsequently stained with mouse anti-IL-6R and rabbit anti-SorLA as primary antibodies and with Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 568-conjugated goat anti-rabbit antibodies as secondary antibodies. Scale bars, 10 μm.
    Figure Legend Snippet: SPR analysis of the binding of sIL-6R to sSorLA and to the Vps10p-D of SorLA (A to D) and SorLA-mediated uptake of sIL-6R in cells (E). The concentration dependence of sIL-6R binding to immobilized sSorLA (A) and the SorLA Vps10p-D (C) was evaluated. The indicated K d values were calculated on the basis of the collected sums of data. The inhibition by SorLA propeptide (pro) of sIL-6R binding to sSorLA (B) and to the Vps10p-D (D) was also evaluated. SorLA was preincubated with a saturating concentration of propeptide (2 μM) prior to the injection of a mixture of propeptide (2 μM) and sIL-6R (100 nM). The response obtained with sIL-6R alone, i.e., the curve to be expected in the absence of inhibition, is indicated in each case in red. (E) Uptake of sIL-6R in SorLA-transfected and wt HEK293 cells. The cells were incubated (37°C, 30 min) at 250 nM sIL-6R in the absence or presence of 20 μM SorLA propeptide. The cells were then washed, fixed, permeabilized, and subsequently stained with mouse anti-IL-6R and rabbit anti-SorLA as primary antibodies and with Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 568-conjugated goat anti-rabbit antibodies as secondary antibodies. Scale bars, 10 μm.

    Techniques Used: SPR Assay, Binding Assay, Concentration Assay, Inhibition, Injection, Transfection, Incubation, Staining

    IL-6 cis signaling but not trans signaling is affected in SorLA transfectants. (A) BA/F3 cells transfected with gp130 and IL-6R alone or in combination with SorLA were incubated (37°C, 15 min) in the absence or presence of 5 nM IL-6. The levels of total and phosphorylated STAT3 were subsequently determined by Western blotting and densitometry. (B and C) HEK293 cells, with endogenous expression of gp130, and transfected with IL-6R alone or in combination with SorLA (B) or SorLAΔtail (C) were stimulated with IL-6 and probed for STAT3 and pSTAT3 as described above. (D) BA/F3 transfected with gp130 or double transfected with gp130 and SorLA were incubated (37°C, 15 min) in blank medium or medium containing both IL-6 and sIL-6R (5 nM each). The cell lysates were analyzed for STAT3 and pSTAT3 as described above. (E) Wild-type HEK293 expressing gp130 and corresponding SorLA transfectants were stimulated and analyzed as in panel D. The left panels (A to E) show Western blot results of single experiments; the right panels sum up the results of several experiments and show results obtained in SorLA transfectants (open columns) relative to values obtained in cells not transfected with SorLA (shaded columns). Each column represents the mean values, and bars indicate the SEM (A, n = 9; B, n = 6; C, n = 6; D, n = 4; E, n = 7). P values were calculated using a Wilcoxon signed-rank test based on raw data. (F) SPR analysis of the binding of IL-6 to immobilized sIL-6R in the presence of a surplus of sSorLA. Soluble IL-6R was subjected to sSorLA (1 μM) prior to the injection of a mixture of sSorLA (1 μM) and IL-6 (100 nM). The response obtained with IL-6 (100 nM) alone is shown.
    Figure Legend Snippet: IL-6 cis signaling but not trans signaling is affected in SorLA transfectants. (A) BA/F3 cells transfected with gp130 and IL-6R alone or in combination with SorLA were incubated (37°C, 15 min) in the absence or presence of 5 nM IL-6. The levels of total and phosphorylated STAT3 were subsequently determined by Western blotting and densitometry. (B and C) HEK293 cells, with endogenous expression of gp130, and transfected with IL-6R alone or in combination with SorLA (B) or SorLAΔtail (C) were stimulated with IL-6 and probed for STAT3 and pSTAT3 as described above. (D) BA/F3 transfected with gp130 or double transfected with gp130 and SorLA were incubated (37°C, 15 min) in blank medium or medium containing both IL-6 and sIL-6R (5 nM each). The cell lysates were analyzed for STAT3 and pSTAT3 as described above. (E) Wild-type HEK293 expressing gp130 and corresponding SorLA transfectants were stimulated and analyzed as in panel D. The left panels (A to E) show Western blot results of single experiments; the right panels sum up the results of several experiments and show results obtained in SorLA transfectants (open columns) relative to values obtained in cells not transfected with SorLA (shaded columns). Each column represents the mean values, and bars indicate the SEM (A, n = 9; B, n = 6; C, n = 6; D, n = 4; E, n = 7). P values were calculated using a Wilcoxon signed-rank test based on raw data. (F) SPR analysis of the binding of IL-6 to immobilized sIL-6R in the presence of a surplus of sSorLA. Soluble IL-6R was subjected to sSorLA (1 μM) prior to the injection of a mixture of sSorLA (1 μM) and IL-6 (100 nM). The response obtained with IL-6 (100 nM) alone is shown.

    Techniques Used: Transfection, Incubation, Western Blot, Expressing, SPR Assay, Binding Assay, Injection

    SorLA accounts for the uptake of sIL-6R in astrocytes. (A) Astrocytes isolated from wt and SorLA ko mice were incubated (37°C, 30 min) in unsupplemented medium or in medium containing 250 nM sIL-6R with or without 20 μM SorLA propeptide (pro) as indicated. The cells were washed, fixed, and permeabilized before staining with mouse anti-IL-6R, rabbit anti-SorLA antibodies, and the matching secondary antibodies. (B) Histogram showing the average number of sIL-6R positive vesicles found in each of nine randomly selected wt and SorLA ko astrocytes. Each column represents the mean value, and bars indicate the SEM. The data were evaluated using one-way ANOVA and Tukey's test. Scale bars, 10 μm.
    Figure Legend Snippet: SorLA accounts for the uptake of sIL-6R in astrocytes. (A) Astrocytes isolated from wt and SorLA ko mice were incubated (37°C, 30 min) in unsupplemented medium or in medium containing 250 nM sIL-6R with or without 20 μM SorLA propeptide (pro) as indicated. The cells were washed, fixed, and permeabilized before staining with mouse anti-IL-6R, rabbit anti-SorLA antibodies, and the matching secondary antibodies. (B) Histogram showing the average number of sIL-6R positive vesicles found in each of nine randomly selected wt and SorLA ko astrocytes. Each column represents the mean value, and bars indicate the SEM. The data were evaluated using one-way ANOVA and Tukey's test. Scale bars, 10 μm.

    Techniques Used: Isolation, Mouse Assay, Incubation, Staining

    Binding (SPR analysis) of IL-6 to sIL-6R, sSorLA, and the Vps10p-D of SorLA (A to E) and SorLA-mediated uptake of IL-6 in cells (F) and in LAMP-1-positive vesicles (G). The concentration dependence of IL-6 binding to immobilized sIL-6R (A), sSorLA (B), and SorLA Vps10p-D (D) was evaluated. IL-6 was applied at the given concentrations, and the indicated K d values were calculated on the basis of the collected sum of data. SorLA propeptide-mediated inhibition of IL-6 (100 nM) binding to sSorLA (C) and the SorLA Vps10p-D (E). SorLA was subjected to saturating concentrations of propeptide (2 μM) prior to injection of a mixture of propeptide (2 μM) and IL-6 (100 nM). The projected red lines indicate IL-6 binding obtained in the absence of propeptide, i.e., curves to be expected if the propeptide does not inhibit. (F) Untransfected and SorLA-transfected HEK293 cells were incubated (37°C, 30 min) at 125 nM IL-6 in the absence or presence of 20 μM SorLA propeptide (pro) as indicated. Subsequently, the cells were washed, fixed, permeabilized, and stained using goat anti-IL-6 and mouse anti-SorLA as primary antibodies, as well as Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated donkey anti-rabbit antibodies as secondary antibodies. (G) Immunofluorescence showing accumulation and LAMP-1 colocalization of IL-6 (arrows) in cells treated with lysosomal inhibitors prior to and during 3 h of incubation with IL-6 (Pearson's r = 0.42). Scale bars, 10 μm.
    Figure Legend Snippet: Binding (SPR analysis) of IL-6 to sIL-6R, sSorLA, and the Vps10p-D of SorLA (A to E) and SorLA-mediated uptake of IL-6 in cells (F) and in LAMP-1-positive vesicles (G). The concentration dependence of IL-6 binding to immobilized sIL-6R (A), sSorLA (B), and SorLA Vps10p-D (D) was evaluated. IL-6 was applied at the given concentrations, and the indicated K d values were calculated on the basis of the collected sum of data. SorLA propeptide-mediated inhibition of IL-6 (100 nM) binding to sSorLA (C) and the SorLA Vps10p-D (E). SorLA was subjected to saturating concentrations of propeptide (2 μM) prior to injection of a mixture of propeptide (2 μM) and IL-6 (100 nM). The projected red lines indicate IL-6 binding obtained in the absence of propeptide, i.e., curves to be expected if the propeptide does not inhibit. (F) Untransfected and SorLA-transfected HEK293 cells were incubated (37°C, 30 min) at 125 nM IL-6 in the absence or presence of 20 μM SorLA propeptide (pro) as indicated. Subsequently, the cells were washed, fixed, permeabilized, and stained using goat anti-IL-6 and mouse anti-SorLA as primary antibodies, as well as Alexa Fluor 488-conjugated donkey anti-goat and Alexa Fluor 568-conjugated donkey anti-rabbit antibodies as secondary antibodies. (G) Immunofluorescence showing accumulation and LAMP-1 colocalization of IL-6 (arrows) in cells treated with lysosomal inhibitors prior to and during 3 h of incubation with IL-6 (Pearson's r = 0.42). Scale bars, 10 μm.

    Techniques Used: Binding Assay, SPR Assay, Concentration Assay, Inhibition, Injection, Transfection, Incubation, Staining, Immunofluorescence

    Soluble SorLA may stabilize IL-6 trans signaling. (A) BA/F3 cells expressing gp130 were stimulated (15 min, 37°C) as indicated, and the resulting levels of pSTAT3/STAT3 were determined by Western blotting of cell lysates. Prior to stimulation, sIL-6R (5 nM) and sSorLA (40 nM) were incubated separately (3 h, room temperature), whereas IL-6 (5 M) was preincubated alone or in combination with 40 nM sSorLA (sSorLA:IL-6). The left panel shows a Western blot of a representative experiment. The right panel summarizes results of several ( n ) experiments in which the pSTAT3 levels (measured by densitometry) were set relative to the level obtained in response to IL-6+IL-6R (assigned the value 1). Bars indicate the SEM, The P value was calculated using the Wilcoxon signed-rank test. (B) The same BA/F3 cells were stimulated (15 min, 37°C) as indicated, but in this case none of the reagents had been coincubated prior to stimulation. The relative pSTAT3 levels were determined as described above. The inset depicts a Western blot of a single experiment, and the histogram summarizes results of nine experiments. (C) pSTAT3 levels in the same cells and stimulated as for panel A except that sSorLA had been substituted with sSorCS3. The inset shows a Western blot of a single experiment, the histogram summarizes (as described above) results of five separate experiments. (D) pSTAT3 levels in SorLA ko astrocytes stimulated with preincubated reagents as for panel A. The columns represent mean values (± SEM, n = 3) relative to the pSTAT3 level in unstimulated astrocytes (assigned value 1). Data were evaluated by using one-way ANOVA and Tukey's test. (E) SPR analysis of the binding of sIL-6R to a preformed sSorLA:IL-6 complex. Immobilized sSorLA was initially exposed to IL-6 (100 nM) prior to the injection of fresh buffer containing 100 nM sIL-6R. The subsequent increase in response units signifies the binding of sIL-6R to the preformed sSorLA:IL-6 complex. The estimated K d is indicated.
    Figure Legend Snippet: Soluble SorLA may stabilize IL-6 trans signaling. (A) BA/F3 cells expressing gp130 were stimulated (15 min, 37°C) as indicated, and the resulting levels of pSTAT3/STAT3 were determined by Western blotting of cell lysates. Prior to stimulation, sIL-6R (5 nM) and sSorLA (40 nM) were incubated separately (3 h, room temperature), whereas IL-6 (5 M) was preincubated alone or in combination with 40 nM sSorLA (sSorLA:IL-6). The left panel shows a Western blot of a representative experiment. The right panel summarizes results of several ( n ) experiments in which the pSTAT3 levels (measured by densitometry) were set relative to the level obtained in response to IL-6+IL-6R (assigned the value 1). Bars indicate the SEM, The P value was calculated using the Wilcoxon signed-rank test. (B) The same BA/F3 cells were stimulated (15 min, 37°C) as indicated, but in this case none of the reagents had been coincubated prior to stimulation. The relative pSTAT3 levels were determined as described above. The inset depicts a Western blot of a single experiment, and the histogram summarizes results of nine experiments. (C) pSTAT3 levels in the same cells and stimulated as for panel A except that sSorLA had been substituted with sSorCS3. The inset shows a Western blot of a single experiment, the histogram summarizes (as described above) results of five separate experiments. (D) pSTAT3 levels in SorLA ko astrocytes stimulated with preincubated reagents as for panel A. The columns represent mean values (± SEM, n = 3) relative to the pSTAT3 level in unstimulated astrocytes (assigned value 1). Data were evaluated by using one-way ANOVA and Tukey's test. (E) SPR analysis of the binding of sIL-6R to a preformed sSorLA:IL-6 complex. Immobilized sSorLA was initially exposed to IL-6 (100 nM) prior to the injection of fresh buffer containing 100 nM sIL-6R. The subsequent increase in response units signifies the binding of sIL-6R to the preformed sSorLA:IL-6 complex. The estimated K d is indicated.

    Techniques Used: Expressing, Western Blot, Incubation, SPR Assay, Binding Assay, Injection

    3) Product Images from "Interleukin 6 Receptor Is an Independent Prognostic Factor and a Potential Therapeutic Target of Ovarian Cancer"

    Article Title: Interleukin 6 Receptor Is an Independent Prognostic Factor and a Potential Therapeutic Target of Ovarian Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118080

    Expression of IL-6 and IL-6R in ovarian cancer cell lines. Real-time RT-PCR of IL-6 (A) and IL-6R (B). Total RNA was collected from seven different ovarian cancer cell lines using TRIzol and subjected to real-time RT-PCR. The 2 -ΔΔCT method was used to calculate the relative abundance with respect to GAPDH expression. Relative fold differences with respect to primary ovarian surface epithelium (OSE) are presented. (C) Western Blot. Cell lysates from seven ovarian cancer cells were resolved by SDS-PAGE and immunoblotted with an antibody against IL-6 and IL-6R. β-Actin was used as a loading control. (D) RT-PCR. RNA was collected and the expressions of full-length IL-6R (IL-6R) and soluble IL-6R (sIL-6R) expression in ovarian cancer cell lines were examined. PCR conditions were as described in Material and Methods. (E) ELISA assay of sIL-6R. Seven ovarian cancer cells (1 x 10 5 each) were plated onto 24-well plates and cultured with 1 ml of serum-free medium for 72 h. Conditioned media were collected and the concentration of human sIL-6R was measured by ELISA. Experiments were repeated three times. n.d.; not detected. (F) Western Blot. Exogenous treatment of IL-6 activates IL-6/IL-6R signaling in ovarian cancer cell lines. SKOV3ip1 cells were stimulated with IL-6 (100 ng/mL) for 30 minutes with or without pretreatment using ranti-IL-6R antibody (1–100 μg/ml) non-immune IgG as control. Cell lysates were collected and equal amount of cell lysates (30 μg) was resolved by 10% SDS-PAGE and immunoblotted with anti–phosphorylated STAT3 (p-STAT3) antibody and anti–phosphorylated p44/42 MAPK (p-ERK1/2) antibody. The membranes were stripped and rehybridized with antibodies detecting the total forms of the protein. Blots are representative of three experiments.
    Figure Legend Snippet: Expression of IL-6 and IL-6R in ovarian cancer cell lines. Real-time RT-PCR of IL-6 (A) and IL-6R (B). Total RNA was collected from seven different ovarian cancer cell lines using TRIzol and subjected to real-time RT-PCR. The 2 -ΔΔCT method was used to calculate the relative abundance with respect to GAPDH expression. Relative fold differences with respect to primary ovarian surface epithelium (OSE) are presented. (C) Western Blot. Cell lysates from seven ovarian cancer cells were resolved by SDS-PAGE and immunoblotted with an antibody against IL-6 and IL-6R. β-Actin was used as a loading control. (D) RT-PCR. RNA was collected and the expressions of full-length IL-6R (IL-6R) and soluble IL-6R (sIL-6R) expression in ovarian cancer cell lines were examined. PCR conditions were as described in Material and Methods. (E) ELISA assay of sIL-6R. Seven ovarian cancer cells (1 x 10 5 each) were plated onto 24-well plates and cultured with 1 ml of serum-free medium for 72 h. Conditioned media were collected and the concentration of human sIL-6R was measured by ELISA. Experiments were repeated three times. n.d.; not detected. (F) Western Blot. Exogenous treatment of IL-6 activates IL-6/IL-6R signaling in ovarian cancer cell lines. SKOV3ip1 cells were stimulated with IL-6 (100 ng/mL) for 30 minutes with or without pretreatment using ranti-IL-6R antibody (1–100 μg/ml) non-immune IgG as control. Cell lysates were collected and equal amount of cell lysates (30 μg) was resolved by 10% SDS-PAGE and immunoblotted with anti–phosphorylated STAT3 (p-STAT3) antibody and anti–phosphorylated p44/42 MAPK (p-ERK1/2) antibody. The membranes were stripped and rehybridized with antibodies detecting the total forms of the protein. Blots are representative of three experiments.

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, SDS Page, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

    Exogenous treatment of IL-6 promotes ovarian cancer cell proliferation, invasion and VEGF production. (A) A matrigel invasion assay was done using a modified Boyden chamber system. 1 x 10 5 of SKOV3ip1 ( left ) or RMUS-S ( right ) cells were placed on the top chamber in serum-free medium and allowed to invade for 72 h. Various concentrations of IL-6 (1–100 ng/ml) or 60 ng/ml of sIL-6R were applied in the bottom chamber as a chemoattractant. 10 μg/ml of anti-IL-6R antibody or non-immune IgG was co-treated. Non-invading cells were removed using a cotton swab, and invading cells on the underside of the filter were enumerated. Relative numbers of invading cells with respect to the control (no IL-6 treatment) are shown. (B) In vitro cell proliferation assay. 1 x 10 4 cells of SKOV3ip1 ( left ) or RMG1 ( right ) cells were plated in 24-well plates in 10% FBS/DMEM for 24 h and then incubated in serum-free medium in the presence or absence of various concentrations of IL-6 (1–100 ng/ml) with or without anti-IL-6R antibody or non-immune IgG as control for 72 h. Cell proliferation was evaluated by a modified MTS assay. Cell proliferation was expressed as the ratio of the number of viable cells. (C) ELISA assay of VEGF-A. 1 x 10 5 SKOV3ip1 cells were plated onto 6-well plates and cultured with 2 ml of serum-free medium in the presence or absence of 100 ng/ml of IL-6 for 72 h. Anti-IL-6R antibody or control IgG was co-treated. Conditioned media were collected and the concentration of human VEGF-A was measured by ELISA. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, *; P
    Figure Legend Snippet: Exogenous treatment of IL-6 promotes ovarian cancer cell proliferation, invasion and VEGF production. (A) A matrigel invasion assay was done using a modified Boyden chamber system. 1 x 10 5 of SKOV3ip1 ( left ) or RMUS-S ( right ) cells were placed on the top chamber in serum-free medium and allowed to invade for 72 h. Various concentrations of IL-6 (1–100 ng/ml) or 60 ng/ml of sIL-6R were applied in the bottom chamber as a chemoattractant. 10 μg/ml of anti-IL-6R antibody or non-immune IgG was co-treated. Non-invading cells were removed using a cotton swab, and invading cells on the underside of the filter were enumerated. Relative numbers of invading cells with respect to the control (no IL-6 treatment) are shown. (B) In vitro cell proliferation assay. 1 x 10 4 cells of SKOV3ip1 ( left ) or RMG1 ( right ) cells were plated in 24-well plates in 10% FBS/DMEM for 24 h and then incubated in serum-free medium in the presence or absence of various concentrations of IL-6 (1–100 ng/ml) with or without anti-IL-6R antibody or non-immune IgG as control for 72 h. Cell proliferation was evaluated by a modified MTS assay. Cell proliferation was expressed as the ratio of the number of viable cells. (C) ELISA assay of VEGF-A. 1 x 10 5 SKOV3ip1 cells were plated onto 6-well plates and cultured with 2 ml of serum-free medium in the presence or absence of 100 ng/ml of IL-6 for 72 h. Anti-IL-6R antibody or control IgG was co-treated. Conditioned media were collected and the concentration of human VEGF-A was measured by ELISA. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, *; P

    Techniques Used: Invasion Assay, Modification, In Vitro, Proliferation Assay, Incubation, MTS Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

    CD11b + CD14 + cells from ovarian cancer ascites promote ovarian cancer cell invasion and proliferation via producing IL-6. (A) The protocol of isolation of CD11b - , CD11b + CD14 - and CD11b + CD14 + cells using magnetic-activated cell sorting (MACS) technology (Miltenyi Biotech). ELISA assay of IL-6 (B) and sIL-6R (C). 1 x 10 5 of SKOV3ip1 cells and primary cells indicated in the figure were plated onto 6-well plates and cultured with 2 ml of serum-free medium for 72 h. Conditioned media were collected and the concentrations of human IL-6 (B) as well as sIL-6R (C) were measured by ELISA. Experiments were repeated three times and values are means (±SD) of triplicates. (D) Matrigel invasion assay. 1 x 10 5 SKOV3ip1 cells were placed on the upper chamber with the same number of primary cells indicated in the figure seeded on the bottom chamber as a chemoattractant, and were allowed to invade for 72 h. The relative number of invading cells when no cells were plated on the bottom chamber was set as 1.0. (E) Anti-IL-6R antibody inhibited ovarian cancer cell invasion induced by CD11b + CD14 + cells. In this experiment, the co-culture experiment in Fig. 4D was repeated with the addition of the 10 μg/ml of anti-IL-6R antibody or non-immune IgG in the bottom chamber. Representative pictures of transwells are shown in Fig. 4D and 4E ( bottom ). (F) In vitro cell proliferation assay. 1 x 10 4 SKOV3ip1 cells were plated in 24-well plates. Thereafter, polycarbonate filters with 1-μm pores were placed onto 24-well plates and the same number of primary cells indicated in the figure were seeded as a stimulant and cells were cultured for 72 h. Cell proliferation was expressed as the ratio of the number of viable cells. (G) Anti-IL-6R antibody inhibited ovarian cancer cell proliferation induced by CD11b + CD14 + cells. In this experiment, the co-culture experiment in Fig. 4F was repeated with the addition of the 10 μg/μl of anti-IL-6R antibody or non-immune IgG in the upper chamber. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, n.d.; not detected, *; P
    Figure Legend Snippet: CD11b + CD14 + cells from ovarian cancer ascites promote ovarian cancer cell invasion and proliferation via producing IL-6. (A) The protocol of isolation of CD11b - , CD11b + CD14 - and CD11b + CD14 + cells using magnetic-activated cell sorting (MACS) technology (Miltenyi Biotech). ELISA assay of IL-6 (B) and sIL-6R (C). 1 x 10 5 of SKOV3ip1 cells and primary cells indicated in the figure were plated onto 6-well plates and cultured with 2 ml of serum-free medium for 72 h. Conditioned media were collected and the concentrations of human IL-6 (B) as well as sIL-6R (C) were measured by ELISA. Experiments were repeated three times and values are means (±SD) of triplicates. (D) Matrigel invasion assay. 1 x 10 5 SKOV3ip1 cells were placed on the upper chamber with the same number of primary cells indicated in the figure seeded on the bottom chamber as a chemoattractant, and were allowed to invade for 72 h. The relative number of invading cells when no cells were plated on the bottom chamber was set as 1.0. (E) Anti-IL-6R antibody inhibited ovarian cancer cell invasion induced by CD11b + CD14 + cells. In this experiment, the co-culture experiment in Fig. 4D was repeated with the addition of the 10 μg/ml of anti-IL-6R antibody or non-immune IgG in the bottom chamber. Representative pictures of transwells are shown in Fig. 4D and 4E ( bottom ). (F) In vitro cell proliferation assay. 1 x 10 4 SKOV3ip1 cells were plated in 24-well plates. Thereafter, polycarbonate filters with 1-μm pores were placed onto 24-well plates and the same number of primary cells indicated in the figure were seeded as a stimulant and cells were cultured for 72 h. Cell proliferation was expressed as the ratio of the number of viable cells. (G) Anti-IL-6R antibody inhibited ovarian cancer cell proliferation induced by CD11b + CD14 + cells. In this experiment, the co-culture experiment in Fig. 4F was repeated with the addition of the 10 μg/μl of anti-IL-6R antibody or non-immune IgG in the upper chamber. Experiments were repeated three times and values are means ± SD of triplicates. n.s.; not significant, n.d.; not detected, *; P

    Techniques Used: Isolation, FACS, Magnetic Cell Separation, Enzyme-linked Immunosorbent Assay, Cell Culture, Invasion Assay, Co-Culture Assay, In Vitro, Proliferation Assay

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    Article Snippet: The transfected cells in FBS-free culture medium were seeded onto the upper chamber while the lower chamber was filled with normal FBS-containing medium. .. For DLL1 stimulation, 2.5 μg of recombinant DLL1 (R & D systems, Minneapolis, MN) was added to the upper chamber. ..

    Article Title: Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes
    Article Snippet: Mouse Fc Block (2.4G2; BD Bioscience, San Jose, CA, USA) and Functional Grade Purified Human Fc(gamma)R-Binding Inhibitor (eBioscience, San Diego, CA, USA) were used to block non-specific binding of mAbs to Fc(gamma) receptors. .. Generation of mAbs To generate the mAbs specific for human Dll1, Dll4, Jagged1 and Jagged2, Balb/c mice (Charles River) were immunized by intraperitoneal injection of human Dll1-Fc or Jagged1-Fc fusion protein, recombinant human Dll4 (R & D Systems), or Jagged2-transfected CHO cells three times at seven-day intervals. ..

    Article Title: Sequential and ?-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth
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    Article Title: Role of IL-22- and TNF-?-Producing Th22 Cells in Uveitis Patients with Beh?et's Disease
    Article Snippet: PBMCs from patients with Behçet’s disease or healthy donors were used to establish CD4+ T cell lines by culturing with anti-human CD3 Ab (2 μg/ml), anti-human CD28 Ab (2 μg/ml), and recombinant human IL-2 (100 U/ml; all from BD Pharmingen) for 5 d. Freshly purified T cells were enriched for CD4+ cells using T cell isolation kits (MACS; Miltenyi Biotec, > 94% CD4+ ) and then used for in vitro assays or flow cytometric analysis. .. For the induction of human Th22 cells, purified CD4+ T cells from Behçet’s disease patients or healthy donors were cocultured with anti-human CD3 Ab (2 μg/ml), anti-human CD28 Ab (2 μg/ml), anti-human IFN-γ Ab (5 μg/ml; R & D Systems), anti-human IL-4 Ab (5 μg/ml; R & D Systems), and recombinant human proteins, such as TNF-α (50 ng/ml; R & D Systems), IL-6 (20 ng/ml; R & D Systems), and rIL-2 (100 U/ml). ..

    Article Title: ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing
    Article Snippet: It can thus allow validation of protein expression patterns in biobank samples and in prospective studies, and the method can provide a platform for clinical use. .. Recombinant proteins and antibodies All recombinant human proteins and all antibodies, with the exception of IgG from mouse serum, were from R & D Systems. .. The IgG from mouse serum was purchased from Sigma-Aldrich.

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    Mouse Assay:

    Article Title: Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes
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    Injection:

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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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    R&D Systems human recombinant soluble il 6 receptor
    Effects of <t>IL-6</t> on capsaicin-evoked CGRP release from adult DRG neurons in culture. A. Adult cultured DRGs were treated with capsaicin (30 nM) alone (i.e.; Control), TB-2-081 (5 μM) followed by capsaicin (TB-2-081), IL-6 (20 ng/ml) followed by
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    R&D Systems recombinant human soluble il 6 receptor
    IL-17 Enhances <t>IL-6-mediated</t> SOCS3 Expression in Primary Astrocytes, and SOCS3 is a Negative Regulator of the Synergistic Effect of IL-6 and IL-17 A , Astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 8 h, and levels of SOCS3 mRNA expression were determined by QRT-PCR. B and C, Astrocytes were transfected with SOCS3 siRNA (100 nM) or siRNA control (100 nM) for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 4 h. Levels of SOCS3 (B) and IL-6 (C) mRNA expression was determined by QRT-PCR. D, Astrocytes were transfected with SOCS3 siRNA or siRNA control for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 24 h, and supernatants analyzed for IL-6 protein by ELISA. E, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were harvested. Digestion of genomic DNA distinguishes the full-length (fl) and excised alleles ( Δ ). F, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, the cells were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h, and levels of IL-6 mRNA expression determined by QRT-PCR. G, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 16 or 24 h, and supernatants analyzed for IL-6 protein by ELISA. All data are the mean ± SD of three experiments. *, p ≤ 0.05;**, p ≤ 0.01.
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    R&D Systems recombinant human sil 6r
    TNF-α interacts with microglia to recruit soluble IL-6 receptor. A , Release of IL-6 soluble receptor (sIL6-R) into the supernatant of BV2 mouse microglia cells within 2 h of stimulation ( n = 6). Basal release (control, 26.0 ± 3.9 pg/ml, mean ± SEM) decreased insignificantly in the presence of minocycline. TNF-α increased <t>sIL-6R</t> release significantly, an effect inhibited by minocycline. There was no additional release of sIL-6R by IL-6. x indicates a significant difference between the indicated groups ( p
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    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

    Effects of IL-6 on capsaicin-evoked CGRP release from adult DRG neurons in culture. A. Adult cultured DRGs were treated with capsaicin (30 nM) alone (i.e.; Control), TB-2-081 (5 μM) followed by capsaicin (TB-2-081), IL-6 (20 ng/ml) followed by

    Journal: Pain

    Article Title: Reversal of pancreatitis-induced pain by an orally available, small molecule interleukin-6 receptor antagonist

    doi: 10.1016/j.pain.2010.05.022

    Figure Lengend Snippet: Effects of IL-6 on capsaicin-evoked CGRP release from adult DRG neurons in culture. A. Adult cultured DRGs were treated with capsaicin (30 nM) alone (i.e.; Control), TB-2-081 (5 μM) followed by capsaicin (TB-2-081), IL-6 (20 ng/ml) followed by

    Article Snippet: Ninety-six well-plates (Nunc) were coated with 500 ng/mL of human recombinant soluble IL-6 receptor (R & D Systems) overnight at 4°C.

    Techniques: Cell Culture

    A. Structure of TB-2-081. B Saturation of IL-6 at human recombinant sIL-6R. Half saturation concentration is 960 pM. C. Competitive binding of TB-2-081 to IL-6R (IC 50 = 29.4 pM). D. TB-2-081 inhibits the growth of IL-6 dependent cell line TF-1(IC 50 =

    Journal: Pain

    Article Title: Reversal of pancreatitis-induced pain by an orally available, small molecule interleukin-6 receptor antagonist

    doi: 10.1016/j.pain.2010.05.022

    Figure Lengend Snippet: A. Structure of TB-2-081. B Saturation of IL-6 at human recombinant sIL-6R. Half saturation concentration is 960 pM. C. Competitive binding of TB-2-081 to IL-6R (IC 50 = 29.4 pM). D. TB-2-081 inhibits the growth of IL-6 dependent cell line TF-1(IC 50 =

    Article Snippet: Ninety-six well-plates (Nunc) were coated with 500 ng/mL of human recombinant soluble IL-6 receptor (R & D Systems) overnight at 4°C.

    Techniques: Recombinant, Concentration Assay, Binding Assay

    Pancreatitis-induced changes in IL-6 expression in the pancreas and DRG. A. Time-course of changes in levels of IL-6 in pancreata of naïve, ethanol vehicle or DBTC-treated animals (*indicates significant difference from naïve group, P

    Journal: Pain

    Article Title: Reversal of pancreatitis-induced pain by an orally available, small molecule interleukin-6 receptor antagonist

    doi: 10.1016/j.pain.2010.05.022

    Figure Lengend Snippet: Pancreatitis-induced changes in IL-6 expression in the pancreas and DRG. A. Time-course of changes in levels of IL-6 in pancreata of naïve, ethanol vehicle or DBTC-treated animals (*indicates significant difference from naïve group, P

    Article Snippet: Ninety-six well-plates (Nunc) were coated with 500 ng/mL of human recombinant soluble IL-6 receptor (R & D Systems) overnight at 4°C.

    Techniques: Expressing

    IL-17 Enhances IL-6-mediated SOCS3 Expression in Primary Astrocytes, and SOCS3 is a Negative Regulator of the Synergistic Effect of IL-6 and IL-17 A , Astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 8 h, and levels of SOCS3 mRNA expression were determined by QRT-PCR. B and C, Astrocytes were transfected with SOCS3 siRNA (100 nM) or siRNA control (100 nM) for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 4 h. Levels of SOCS3 (B) and IL-6 (C) mRNA expression was determined by QRT-PCR. D, Astrocytes were transfected with SOCS3 siRNA or siRNA control for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 24 h, and supernatants analyzed for IL-6 protein by ELISA. E, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were harvested. Digestion of genomic DNA distinguishes the full-length (fl) and excised alleles ( Δ ). F, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, the cells were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h, and levels of IL-6 mRNA expression determined by QRT-PCR. G, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 16 or 24 h, and supernatants analyzed for IL-6 protein by ELISA. All data are the mean ± SD of three experiments. *, p ≤ 0.05;**, p ≤ 0.01.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

    doi: 10.4049/jimmunol.1000142

    Figure Lengend Snippet: IL-17 Enhances IL-6-mediated SOCS3 Expression in Primary Astrocytes, and SOCS3 is a Negative Regulator of the Synergistic Effect of IL-6 and IL-17 A , Astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 8 h, and levels of SOCS3 mRNA expression were determined by QRT-PCR. B and C, Astrocytes were transfected with SOCS3 siRNA (100 nM) or siRNA control (100 nM) for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 4 h. Levels of SOCS3 (B) and IL-6 (C) mRNA expression was determined by QRT-PCR. D, Astrocytes were transfected with SOCS3 siRNA or siRNA control for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 24 h, and supernatants analyzed for IL-6 protein by ELISA. E, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were harvested. Digestion of genomic DNA distinguishes the full-length (fl) and excised alleles ( Δ ). F, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, the cells were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h, and levels of IL-6 mRNA expression determined by QRT-PCR. G, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 16 or 24 h, and supernatants analyzed for IL-6 protein by ELISA. All data are the mean ± SD of three experiments. *, p ≤ 0.05;**, p ≤ 0.01.

    Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Infection, Incubation

    Proposed Model of IL-17 Enhancement of the IL-6 Signaling Cascade in Astrocytes A, IL-6/R and IL-17 activate the NF-κB and MAPK pathway, which then induce IL-6 gene expression. The synergistic effect of these two mediators depends on NF-κB, p38 and JNK MAPK activity. Activated NF-κB p65, c-Fos and c-Jun bind to the IL-6 promoter. Concurrent with NF-κB and MAPK recruitment, IL-6/R and IL-17 leads to the recruitment of coactivators CBP and p300, modifications in AcH3 and AcH4, and recruitment of RNA Pol II to the IL-6 promoter, which results in transcriptional activation of the IL-6 gene. IL-17 can also enhance IL-6/R induced SOCS3 expression, and SOCS3 inhibits IL-6/R plus IL-17-induced NF-κB and MAPK activation, which results in a reduction of IL-6 gene expression in astrocytes. B , Naive CD4 + T cells, after activation by signaling through the T cell receptor and co-stimulatory molecules, can differentiate into Th17 cells in the presence of IL-6, TGF-β, IL-1 and IL-23. IL-17 together with IL-6/R triggers a positive-feedforward loop of IL-6 expression in astrocytes, which may also influence Th17 cell differentiation. SOCS3 participates in these processes as a negative feedback regulator. See text for details.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

    doi: 10.4049/jimmunol.1000142

    Figure Lengend Snippet: Proposed Model of IL-17 Enhancement of the IL-6 Signaling Cascade in Astrocytes A, IL-6/R and IL-17 activate the NF-κB and MAPK pathway, which then induce IL-6 gene expression. The synergistic effect of these two mediators depends on NF-κB, p38 and JNK MAPK activity. Activated NF-κB p65, c-Fos and c-Jun bind to the IL-6 promoter. Concurrent with NF-κB and MAPK recruitment, IL-6/R and IL-17 leads to the recruitment of coactivators CBP and p300, modifications in AcH3 and AcH4, and recruitment of RNA Pol II to the IL-6 promoter, which results in transcriptional activation of the IL-6 gene. IL-17 can also enhance IL-6/R induced SOCS3 expression, and SOCS3 inhibits IL-6/R plus IL-17-induced NF-κB and MAPK activation, which results in a reduction of IL-6 gene expression in astrocytes. B , Naive CD4 + T cells, after activation by signaling through the T cell receptor and co-stimulatory molecules, can differentiate into Th17 cells in the presence of IL-6, TGF-β, IL-1 and IL-23. IL-17 together with IL-6/R triggers a positive-feedforward loop of IL-6 expression in astrocytes, which may also influence Th17 cell differentiation. SOCS3 participates in these processes as a negative feedback regulator. See text for details.

    Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Activity Assay, Activation Assay, Cell Differentiation

    IL-17 Synergizes with IL-6/R for Activation of the NF-κB and MAPK Pathways A , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against IκBα and GAPDH. The basal level of the untreated sample was set at 100, and the percentage change of IκBα upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment compared with the basal value. B , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65 and GAPDH. C , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against phospho-ERK1/2, ERK1/2 and GAPDH. D , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R and IL-17 for 30 min and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p38, p38 and GAPDH. E , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-JNK, JNK and GAPDH. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment was compared with that value ( B , C , D and E ). Representative of at least three experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

    doi: 10.4049/jimmunol.1000142

    Figure Lengend Snippet: IL-17 Synergizes with IL-6/R for Activation of the NF-κB and MAPK Pathways A , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against IκBα and GAPDH. The basal level of the untreated sample was set at 100, and the percentage change of IκBα upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment compared with the basal value. B , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65 and GAPDH. C , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against phospho-ERK1/2, ERK1/2 and GAPDH. D , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R and IL-17 for 30 min and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p38, p38 and GAPDH. E , Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-JNK, JNK and GAPDH. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment was compared with that value ( B , C , D and E ). Representative of at least three experiments.

    Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R & D Systems (Minneapolis, MN).

    Techniques: Activation Assay, Incubation

    IL-17 and IL-6/R Induction of IL-6 Depends on NF-κB, p38 and JNK MAPK Activity A , Astrocytes were cultured in absence or presence of IL-17 followed by IL-6/R treatment for 4 h. Actinomycin D (5 ng/ml) was then added, and cells harvested at 0, 30, 60, 120, and 240 min after addition. The abundance of IL-6 mRNA was determined by QRT-PCR. B , C , DMSO vehicle, BAY 11 (5 μM), U0126 (10 μM), SB203580 (10 μM) or JNKi II (10 μM) were added to cultures 1 h before cytokine addition, and then astrocytes were incubated with medium, IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h. Levels of IL-6 mRNA expression were determined by QRT-PCR. All data are the mean ± SD of three experiments. **, p ≤ 0.01; NS = not significant.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

    doi: 10.4049/jimmunol.1000142

    Figure Lengend Snippet: IL-17 and IL-6/R Induction of IL-6 Depends on NF-κB, p38 and JNK MAPK Activity A , Astrocytes were cultured in absence or presence of IL-17 followed by IL-6/R treatment for 4 h. Actinomycin D (5 ng/ml) was then added, and cells harvested at 0, 30, 60, 120, and 240 min after addition. The abundance of IL-6 mRNA was determined by QRT-PCR. B , C , DMSO vehicle, BAY 11 (5 μM), U0126 (10 μM), SB203580 (10 μM) or JNKi II (10 μM) were added to cultures 1 h before cytokine addition, and then astrocytes were incubated with medium, IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h. Levels of IL-6 mRNA expression were determined by QRT-PCR. All data are the mean ± SD of three experiments. **, p ≤ 0.01; NS = not significant.

    Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R & D Systems (Minneapolis, MN).

    Techniques: Activity Assay, Cell Culture, Quantitative RT-PCR, Incubation, Expressing

    IL-6/R plus IL-17 Enhances Recruitment of p65, P-p65, c-Jun, c-Fos, CBP, p300, and RNA Pol II to the IL-6 Promoter A, Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 90 min, and then cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with Abs against p65, P-p65, c-Fos, c-Jun, or normal rabbit IgG. PCR analysis of the positive control (input) indicates that soluble chromatin samples obtained from each time point had equal amounts of chromatin fragments containing the IL-6 promoter. B, Primary astrocytes were treated as above. Soluble chromatin was subjected to immunoprecipitation with Abs against histone acetylation (Ac-H3 and Ac-H4), p300, CBP, RNA Pol II or normal rabbit IgG. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R plus IL-17 treatment was compared with that value. Representative of at least three experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

    doi: 10.4049/jimmunol.1000142

    Figure Lengend Snippet: IL-6/R plus IL-17 Enhances Recruitment of p65, P-p65, c-Jun, c-Fos, CBP, p300, and RNA Pol II to the IL-6 Promoter A, Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 90 min, and then cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with Abs against p65, P-p65, c-Fos, c-Jun, or normal rabbit IgG. PCR analysis of the positive control (input) indicates that soluble chromatin samples obtained from each time point had equal amounts of chromatin fragments containing the IL-6 promoter. B, Primary astrocytes were treated as above. Soluble chromatin was subjected to immunoprecipitation with Abs against histone acetylation (Ac-H3 and Ac-H4), p300, CBP, RNA Pol II or normal rabbit IgG. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R plus IL-17 treatment was compared with that value. Representative of at least three experiments.

    Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R & D Systems (Minneapolis, MN).

    Techniques: Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Activation Assay

    Enhanced Activation of NF-κB and MAPK Pathways in SOCS3 Deficient Astrocytes To evaluate NF-κB and MAPK activation in the absence or presence of SOCS3, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were treated with medium (UN) or IL-6/R plus IL-17 for 15, 30, 60 or 120 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65, phospho-p38, p38, phospho-JNK, JNK or GAPDH. The basal level of the untreated GFP infected sample was set at 1.0 and fold activation upon IL-6/R plus IL-17 treatment compared with that value. Representative of at least three experiments.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

    doi: 10.4049/jimmunol.1000142

    Figure Lengend Snippet: Enhanced Activation of NF-κB and MAPK Pathways in SOCS3 Deficient Astrocytes To evaluate NF-κB and MAPK activation in the absence or presence of SOCS3, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were treated with medium (UN) or IL-6/R plus IL-17 for 15, 30, 60 or 120 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65, phospho-p38, p38, phospho-JNK, JNK or GAPDH. The basal level of the untreated GFP infected sample was set at 1.0 and fold activation upon IL-6/R plus IL-17 treatment compared with that value. Representative of at least three experiments.

    Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R & D Systems (Minneapolis, MN).

    Techniques: Activation Assay, Infection

    IL-17 Enhances IL-6/sIL-6R-mediated IL-6 Expression in Primary Astrocytes A , IL-17RA and IL-17RC mRNA expression was determined by RT-PCR in RAW264.7 cells (positive control), and primary astrocytes, in duplicate. B , Primary astrocytes were treated with medium (UN), IL-6/R (IL-6, 10 ng/ml and sIL-6R, 25 ng/ml), IL-17 (25 ng/ml) or IL-6/R plus IL-17 for up to 24 h, and levels of IL-6 and GAPDH mRNA expression were determined by RT-PCR and QRT-PCR. C , Primary astrocytes were treated with medium (UN), IL-6/R, different concentrations of IL-17 (1-50 ng/ml) or IL-6/R + IL-17 (1-50 ng/ml) for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. D , Primary astrocytes were treated with medium (UN), IL-6, sIL-6R or IL-17 in various combinations for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. E , Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 48 h, and supernatants were analyzed for IL-6 protein using ELISA. Experiments shown are representative of at least three experiments. All data are the mean ± SD of three experiments. *, p ≤0.05; **, p ≤0.01.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

    doi: 10.4049/jimmunol.1000142

    Figure Lengend Snippet: IL-17 Enhances IL-6/sIL-6R-mediated IL-6 Expression in Primary Astrocytes A , IL-17RA and IL-17RC mRNA expression was determined by RT-PCR in RAW264.7 cells (positive control), and primary astrocytes, in duplicate. B , Primary astrocytes were treated with medium (UN), IL-6/R (IL-6, 10 ng/ml and sIL-6R, 25 ng/ml), IL-17 (25 ng/ml) or IL-6/R plus IL-17 for up to 24 h, and levels of IL-6 and GAPDH mRNA expression were determined by RT-PCR and QRT-PCR. C , Primary astrocytes were treated with medium (UN), IL-6/R, different concentrations of IL-17 (1-50 ng/ml) or IL-6/R + IL-17 (1-50 ng/ml) for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. D , Primary astrocytes were treated with medium (UN), IL-6, sIL-6R or IL-17 in various combinations for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. E , Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 48 h, and supernatants were analyzed for IL-6 protein using ELISA. Experiments shown are representative of at least three experiments. All data are the mean ± SD of three experiments. *, p ≤0.05; **, p ≤0.01.

    Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R & D Systems (Minneapolis, MN).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    TNF-α interacts with microglia to recruit soluble IL-6 receptor. A , Release of IL-6 soluble receptor (sIL6-R) into the supernatant of BV2 mouse microglia cells within 2 h of stimulation ( n = 6). Basal release (control, 26.0 ± 3.9 pg/ml, mean ± SEM) decreased insignificantly in the presence of minocycline. TNF-α increased sIL-6R release significantly, an effect inhibited by minocycline. There was no additional release of sIL-6R by IL-6. x indicates a significant difference between the indicated groups ( p

    Journal: The Journal of Neuroscience

    Article Title: Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha

    doi: 10.1523/JNEUROSCI.4159-15.2016

    Figure Lengend Snippet: TNF-α interacts with microglia to recruit soluble IL-6 receptor. A , Release of IL-6 soluble receptor (sIL6-R) into the supernatant of BV2 mouse microglia cells within 2 h of stimulation ( n = 6). Basal release (control, 26.0 ± 3.9 pg/ml, mean ± SEM) decreased insignificantly in the presence of minocycline. TNF-α increased sIL-6R release significantly, an effect inhibited by minocycline. There was no additional release of sIL-6R by IL-6. x indicates a significant difference between the indicated groups ( p

    Article Snippet: According to experimental protocols, recombinant rat TNF-α (ImmunoTools), recombinant rat IL-6 (Life Technologies), recombinant sgp130, recombinant human IL-6, and/or recombinant human sIL-6R (R & D Systems) were then applied directly in Tyrode's solution onto the spinal cord surface into the trough over the recording site at a concentration of 1 μg/ml.

    Techniques:

    Effect of minocycline on TNF-α-induced hyperexcitability. A , There were no changes of neuronal responses to mechanical stimulation after application of minocycline only and no increase after subsequent application of minocycline plus TNF-α ( n = 11), but there was an increase of the responses after TNF-α plus sIL-6R in the presence of minocycline ( n = 7). Data are shown as the mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the minocycline/TNF-α group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the minocycline/TNF-α/sIL-6R group: innocuous pressure knee 42.3 ± 28.2, noxious pressure knee 326.1 ± 98.7, innocuous pressure ankle 112.3 ± 60.3, noxious pressure ankle 554.9 ± 160.5. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p

    Journal: The Journal of Neuroscience

    Article Title: Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha

    doi: 10.1523/JNEUROSCI.4159-15.2016

    Figure Lengend Snippet: Effect of minocycline on TNF-α-induced hyperexcitability. A , There were no changes of neuronal responses to mechanical stimulation after application of minocycline only and no increase after subsequent application of minocycline plus TNF-α ( n = 11), but there was an increase of the responses after TNF-α plus sIL-6R in the presence of minocycline ( n = 7). Data are shown as the mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the minocycline/TNF-α group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the minocycline/TNF-α/sIL-6R group: innocuous pressure knee 42.3 ± 28.2, noxious pressure knee 326.1 ± 98.7, innocuous pressure ankle 112.3 ± 60.3, noxious pressure ankle 554.9 ± 160.5. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p

    Article Snippet: According to experimental protocols, recombinant rat TNF-α (ImmunoTools), recombinant rat IL-6 (Life Technologies), recombinant sgp130, recombinant human IL-6, and/or recombinant human sIL-6R (R & D Systems) were then applied directly in Tyrode's solution onto the spinal cord surface into the trough over the recording site at a concentration of 1 μg/ml.

    Techniques:

    Increase of spinal IL-6 release during peripheral stimulation in the presence of spinally applied TNF-α and neuronal effects of IL-6 and IL-6/sIL-6R. A , Spinal IL-6 release (means ± SEM) into the spinal cord supernatant in the course of the stimulation protocol and spinal TNF-α application. Samples were extracted after 1 h of baseline (control) and 1 and 2 h after TNF-α application ( n = 10). *Significant increase after TNF-α compared with control before TNF-α ( p

    Journal: The Journal of Neuroscience

    Article Title: Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha

    doi: 10.1523/JNEUROSCI.4159-15.2016

    Figure Lengend Snippet: Increase of spinal IL-6 release during peripheral stimulation in the presence of spinally applied TNF-α and neuronal effects of IL-6 and IL-6/sIL-6R. A , Spinal IL-6 release (means ± SEM) into the spinal cord supernatant in the course of the stimulation protocol and spinal TNF-α application. Samples were extracted after 1 h of baseline (control) and 1 and 2 h after TNF-α application ( n = 10). *Significant increase after TNF-α compared with control before TNF-α ( p

    Article Snippet: According to experimental protocols, recombinant rat TNF-α (ImmunoTools), recombinant rat IL-6 (Life Technologies), recombinant sgp130, recombinant human IL-6, and/or recombinant human sIL-6R (R & D Systems) were then applied directly in Tyrode's solution onto the spinal cord surface into the trough over the recording site at a concentration of 1 μg/ml.

    Techniques:

    Model of the cooperation of TNF-α and IL-6 in the generation of spinal hyperexcitability. TNF-α leads to IL-6 release mainly from neurons and acts on microglia to provide sIL-6R for IL-6 trans -signaling by ADAM10/17-protease-dependent cleavage of IL-6R, which is not present in neurons. Interaction of IL-6-bound IL-6R or IL-6/sIL-6R complex with ubiquitously expressed and homodimerized gp130 is required for IL-6 signaling.

    Journal: The Journal of Neuroscience

    Article Title: Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha

    doi: 10.1523/JNEUROSCI.4159-15.2016

    Figure Lengend Snippet: Model of the cooperation of TNF-α and IL-6 in the generation of spinal hyperexcitability. TNF-α leads to IL-6 release mainly from neurons and acts on microglia to provide sIL-6R for IL-6 trans -signaling by ADAM10/17-protease-dependent cleavage of IL-6R, which is not present in neurons. Interaction of IL-6-bound IL-6R or IL-6/sIL-6R complex with ubiquitously expressed and homodimerized gp130 is required for IL-6 signaling.

    Article Snippet: According to experimental protocols, recombinant rat TNF-α (ImmunoTools), recombinant rat IL-6 (Life Technologies), recombinant sgp130, recombinant human IL-6, and/or recombinant human sIL-6R (R & D Systems) were then applied directly in Tyrode's solution onto the spinal cord surface into the trough over the recording site at a concentration of 1 μg/ml.

    Techniques:

    Induction of hyperexcitability of deep dorsal horn neurons by TNF-α via IL-6/sIL-6R. A , Hypothetical flow chart of an upstream function of TNF-α initiating the release of IL-6 and sIL-6R from different sources to form the downstream IL-6/sIL-6R complex for IL-6 trans -signaling. Indicated are the action sites of specific inhibitors. B , Increases of neuronal responses to mechanical stimulation by spinal application of IL-6/sIL-6R ( n = 6) and spinal application of IL-6/sIL-6R plus etanercept ( n = 6) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the IL-6/sIL-6R group: innocuous pressure knee 154.5 ± 23.2, noxious pressure knee 357.8 ± 43.1, innocuous pressure ankle 67.0 ± 28.7, noxious pressure ankle 470.4 ± 80.7. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus etanercept group: innocuous pressure knee 81.6 ± 47.8, noxious pressure knee 298.3 ± 84.6, innocuous pressure ankle 53.8 ± 44.0, noxious pressure ankle 322.7 ± 135.4. C , Increases of neuronal responses to mechanical stimulation by spinal application of TNF-α plus minocycline ( n = 11) or IL-6/sIL-6R plus minocycline ( n = 8) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the TNF-α plus minocycline group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus minocycline group: innocuous pressure knee 98.4 ± 34.4, noxious pressure knee 292.5 ± 53.1, innocuous pressure ankle 56.4 ± 36.1, noxious pressure ankle 261.2 ± 54.4. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p

    Journal: The Journal of Neuroscience

    Article Title: Involvement of Spinal IL-6 Trans-Signaling in the Induction of Hyperexcitability of Deep Dorsal Horn Neurons by Spinal Tumor Necrosis Factor-Alpha

    doi: 10.1523/JNEUROSCI.4159-15.2016

    Figure Lengend Snippet: Induction of hyperexcitability of deep dorsal horn neurons by TNF-α via IL-6/sIL-6R. A , Hypothetical flow chart of an upstream function of TNF-α initiating the release of IL-6 and sIL-6R from different sources to form the downstream IL-6/sIL-6R complex for IL-6 trans -signaling. Indicated are the action sites of specific inhibitors. B , Increases of neuronal responses to mechanical stimulation by spinal application of IL-6/sIL-6R ( n = 6) and spinal application of IL-6/sIL-6R plus etanercept ( n = 6) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the IL-6/sIL-6R group: innocuous pressure knee 154.5 ± 23.2, noxious pressure knee 357.8 ± 43.1, innocuous pressure ankle 67.0 ± 28.7, noxious pressure ankle 470.4 ± 80.7. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus etanercept group: innocuous pressure knee 81.6 ± 47.8, noxious pressure knee 298.3 ± 84.6, innocuous pressure ankle 53.8 ± 44.0, noxious pressure ankle 322.7 ± 135.4. C , Increases of neuronal responses to mechanical stimulation by spinal application of TNF-α plus minocycline ( n = 11) or IL-6/sIL-6R plus minocycline ( n = 8) for 2 h, shown as mean number of action potentials per 15 s (mean APs/15 s) ± SEM for intervals of 30 min. Baseline values (in APs/15 s) in the TNF-α plus minocycline group: innocuous pressure knee 288.3 ± 105.2, noxious pressure knee 636.8 ± 113.0, innocuous pressure ankle 260.9 ± 69.8, noxious pressure ankle 482.9 ± 84.8. Baseline values (in APs/15 s) in the IL-6/sIL-6R plus minocycline group: innocuous pressure knee 98.4 ± 34.4, noxious pressure knee 292.5 ± 53.1, innocuous pressure ankle 56.4 ± 36.1, noxious pressure ankle 261.2 ± 54.4. Asterisks with arrow indicate the start of significant increases of responses or significant increase of responses at the specific time point ( p

    Article Snippet: According to experimental protocols, recombinant rat TNF-α (ImmunoTools), recombinant rat IL-6 (Life Technologies), recombinant sgp130, recombinant human IL-6, and/or recombinant human sIL-6R (R & D Systems) were then applied directly in Tyrode's solution onto the spinal cord surface into the trough over the recording site at a concentration of 1 μg/ml.

    Techniques: Flow Cytometry