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R&D Systems biotinylated goat anti mouse il 18r antibody
FIGURE 1. <t>IL-18R</t> in the three MEF strains. MEF cells grown for 2 days were stained against mouse IL-18R chain and visualized using confocal digital microscopy. Red dye, anti-IL-18R; blue dye, nuclei; green dye, cell membranes (the last only shown in insets). A, WT; B, IL-18 ko; C, IL-18R ko. The insets show each strain stained with an IgG control instead of the anti-IL-18R antibody.

FIGURE 1. <t>IL-18R</t> in the three MEF strains. MEF cells grown for 2 days were stained against mouse IL-18R chain and visualized using confocal digital microscopy. Red dye, anti-IL-18R; blue dye, nuclei; green dye, cell membranes (the last only shown in insets). A, WT; B, IL-18 ko; C, IL-18R ko. The insets show each strain stained with an IgG control instead of the anti-IL-18R antibody.

Biotinylated Goat Anti Mouse Il 18r Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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biotinylated goat anti mouse il 18r antibody - by Bioz Stars, 2026-09

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1) Product Images from "Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling"

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

Journal: Journal of Biological Chemistry

doi: 10.1074/jbc.m109.004184

FIGURE 1. IL-18R in the three MEF strains. MEF cells grown for 2 days were stained against mouse IL-18R chain and visualized using confocal digital microscopy. Red dye, anti-IL-18R; blue dye, nuclei; green dye, cell membranes (the last only shown in insets). A, WT; B, IL-18 ko; C, IL-18R ko. The insets show each strain stained with an IgG control instead of the anti-IL-18R antibody.

Figure Legend Snippet: FIGURE 1. IL-18R in the three MEF strains. MEF cells grown for 2 days were stained against mouse IL-18R chain and visualized using confocal digital microscopy. Red dye, anti-IL-18R; blue dye, nuclei; green dye, cell membranes (the last only shown in insets). A, WT; B, IL-18 ko; C, IL-18R ko. The insets show each strain stained with an IgG control instead of the anti-IL-18R antibody.

Techniques Used: Staining, Microscopy, Control

FIGURE 2. Production of MIP-1 in IL-1- and TNF-stimulated MEF cells. WT, IL-18 ko, and IL-18R ko MEF cells were stimulated with the indicated concentrations of either IL-1 or TNF, and after 20 h (A, B, and D) or 6 h (C; 25 ng/ml IL-1), the levels of MIP-1 were measured in the supernatants. Results represent MIP-1 in pg/mg total protein in IL-1-exposed (A, C, and D) or TNF-exposed (B and D) cells. The data are means S.E. of four independent experiments, each experiment performed on a separate cell line derived from a different mother (see Table 1). A–C, *, p 0.05; ***, p 0.001 compared with the values in WT cells. D, comparison of IL-1 (1 ng/ml) with TNF (10 ng/ml) as stimuli of MEF cells. Data are means S.E. (n 4); *, p 0.05; ***, p 0.001 for IL-1- compared with TNF-stimulated MEF.

Figure Legend Snippet: FIGURE 2. Production of MIP-1 in IL-1- and TNF-stimulated MEF cells. WT, IL-18 ko, and IL-18R ko MEF cells were stimulated with the indicated concentrations of either IL-1 or TNF, and after 20 h (A, B, and D) or 6 h (C; 25 ng/ml IL-1), the levels of MIP-1 were measured in the supernatants. Results represent MIP-1 in pg/mg total protein in IL-1-exposed (A, C, and D) or TNF-exposed (B and D) cells. The data are means S.E. of four independent experiments, each experiment performed on a separate cell line derived from a different mother (see Table 1). A–C, *, p 0.05; ***, p 0.001 compared with the values in WT cells. D, comparison of IL-1 (1 ng/ml) with TNF (10 ng/ml) as stimuli of MEF cells. Data are means S.E. (n 4); *, p 0.05; ***, p 0.001 for IL-1- compared with TNF-stimulated MEF.

Techniques Used: Derivative Assay, Comparison

FIGURE 3. Differential induction of IL-1, IL-6, and PGE2 in the MEF cell strains. WT, IL-18 ko, and IL-18R ko were stimulated with IL-1 or TNF. After 20 h, supernatants were removed and used to generate D and E and the data shown in Fig. 2. The cells were lysed, and levels of IL-1 were measured. The graphs showmeansofnormalizedcytokineorprostaglandinconcentrationsS.E.(n4).A,B,D,andE,*,p0.05;**,p0.01;***,p0.001comparingthekostrains with WT cells. u and v, values in wild type (u) and/or IL-18 ko MEF (v) were below the detection levels of the ECL. A and D, note the breaks in IL-18R bars. E, #, p 0.05; ##, p 0.01 for IL-18 ko versus IL-18R ko. C, 1 ng/ml IL-1 versus 10 ng/ml TNF. The data are mean S.E. of the same four independent experiments as in Fig. 2D. *, p 0.05 for IL-1-stimulated compared with TNF-stimulated cells.

Figure Legend Snippet: FIGURE 3. Differential induction of IL-1, IL-6, and PGE2 in the MEF cell strains. WT, IL-18 ko, and IL-18R ko were stimulated with IL-1 or TNF. After 20 h, supernatants were removed and used to generate D and E and the data shown in Fig. 2. The cells were lysed, and levels of IL-1 were measured. The graphs showmeansofnormalizedcytokineorprostaglandinconcentrationsS.E.(n4).A,B,D,andE,*,p0.05;**,p0.01;***,p0.001comparingthekostrains with WT cells. u and v, values in wild type (u) and/or IL-18 ko MEF (v) were below the detection levels of the ECL. A and D, note the breaks in IL-18R bars. E, #, p 0.05; ##, p 0.01 for IL-18 ko versus IL-18R ko. C, 1 ng/ml IL-1 versus 10 ng/ml TNF. The data are mean S.E. of the same four independent experiments as in Fig. 2D. *, p 0.05 for IL-1-stimulated compared with TNF-stimulated cells.

Techniques Used:

FIGURE 4. Effect of an anti-IL-18R blocking antibody on cytokine production. Increasing concentrations of an anti-IL-18R monoclonal blocking antibody (shown below the horizontal axis in ng/ml) were added to WT or IL-18 ko MEF cells. After 20 h of incubation, the levels of spontaneous IL-6 were measured in the supernatant. A, the level of IL-6 in the absence of anti-IL-18R was set at 1.0 for each of three independent experiments, and the -fold increase was calculated. The data are mean S.E. -fold increase. *, p 0.05; **, p 0.01; ***, p 0.001 compared with in the absence of the antibody. B, an experiment similar to A, except IL-1 (1 ng/ml) was added to the cells 20 min after the anti-IL-18R antibody. The data in C and D are from the same cultures as shown in A and B. In C and D, MIP-1 was measured in the supernatants. **, p 0.01; ***, p 0.001 compared with no antibody.

Figure Legend Snippet: FIGURE 4. Effect of an anti-IL-18R blocking antibody on cytokine production. Increasing concentrations of an anti-IL-18R monoclonal blocking antibody (shown below the horizontal axis in ng/ml) were added to WT or IL-18 ko MEF cells. After 20 h of incubation, the levels of spontaneous IL-6 were measured in the supernatant. A, the level of IL-6 in the absence of anti-IL-18R was set at 1.0 for each of three independent experiments, and the -fold increase was calculated. The data are mean S.E. -fold increase. *, p 0.05; **, p 0.01; ***, p 0.001 compared with in the absence of the antibody. B, an experiment similar to A, except IL-1 (1 ng/ml) was added to the cells 20 min after the anti-IL-18R antibody. The data in C and D are from the same cultures as shown in A and B. In C and D, MIP-1 was measured in the supernatants. **, p 0.01; ***, p 0.001 compared with no antibody.

Techniques Used: Blocking Assay, Incubation

FIGURE 5. Silencing of the IL-18R in A549 lung epithelial cells. Using shRNA, stable A549 cell lines depleted of the IL-18R -chain were created. For control, A549 cells were transfected with scrambled shRNA. The knockdown of the IL-18R chain was assessed by Western blotting (A) and real time PCR (B). A, the arrows indicate the clones used in the experiments. C, the three scrambled-transfected and two shIL-18R-transfected cell lines shown in A and B were stimulated with 10 ng/ml IL-1 or were left untreated for 20 h. Thereafter, IL-6 levels were determined. The panel depicts means of normal- ized cytokine concentrations S.E.; n 6 clones times 4 independent exper- iments; ***, p 0.001 for shIL-18R versus small hairpin scrambled (shscrambled).

Figure Legend Snippet: FIGURE 5. Silencing of the IL-18R in A549 lung epithelial cells. Using shRNA, stable A549 cell lines depleted of the IL-18R -chain were created. For control, A549 cells were transfected with scrambled shRNA. The knockdown of the IL-18R chain was assessed by Western blotting (A) and real time PCR (B). A, the arrows indicate the clones used in the experiments. C, the three scrambled-transfected and two shIL-18R-transfected cell lines shown in A and B were stimulated with 10 ng/ml IL-1 or were left untreated for 20 h. Thereafter, IL-6 levels were determined. The panel depicts means of normal- ized cytokine concentrations S.E.; n 6 clones times 4 independent exper- iments; ***, p 0.001 for shIL-18R versus small hairpin scrambled (shscrambled).

Techniques Used: shRNA, Control, Transfection, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Clone Assay

FIGURE6.EffectsofibuprofenandIL-1RaontheproductionofIL-6inMEF cells. A, constitutive secretion of IL-6 was measured in the absence or pres- ence of the IL-1Ra. IL-1Ra was added to the cultures of IL-18R ko cells at 10 g/ml, and after 20 h, levels of IL-6 were measured. In cultures without IL-1Ra (), the level of IL-6 was set at 100%, and the mean percentage change of parallel cultures with IL-1Ra was calculated. Data are mean S.E. percentage change from three independent experiments. ***, p 0.001 compared with cultures without IL-1Ra. B, constitutive production of IL-6 in WT, IL-18 ko, and IL-18R ko MEF cells in the absence and presence of ibuprofen (10 M) after 20 h of incubation. C, each of the MEF strains were stimulated with IL-1 (1 ng/ml) in the absence or presence of ibuprofen (ibu; 10 M). After 20 h, levels of IL-6 were measured in the supernatants. Note the break in the IL-18R bar. B and C,thedataaremeanS.E.ofthreeindependentexperiments.*,p0.05;**,p 0.01; ***, p 0.001 comparing cultures with ibuprofen with those without.

Figure Legend Snippet: FIGURE6.EffectsofibuprofenandIL-1RaontheproductionofIL-6inMEF cells. A, constitutive secretion of IL-6 was measured in the absence or pres- ence of the IL-1Ra. IL-1Ra was added to the cultures of IL-18R ko cells at 10 g/ml, and after 20 h, levels of IL-6 were measured. In cultures without IL-1Ra (), the level of IL-6 was set at 100%, and the mean percentage change of parallel cultures with IL-1Ra was calculated. Data are mean S.E. percentage change from three independent experiments. ***, p 0.001 compared with cultures without IL-1Ra. B, constitutive production of IL-6 in WT, IL-18 ko, and IL-18R ko MEF cells in the absence and presence of ibuprofen (10 M) after 20 h of incubation. C, each of the MEF strains were stimulated with IL-1 (1 ng/ml) in the absence or presence of ibuprofen (ibu; 10 M). After 20 h, levels of IL-6 were measured in the supernatants. Note the break in the IL-18R bar. B and C,thedataaremeanS.E.ofthreeindependentexperiments.*,p0.05;**,p 0.01; ***, p 0.001 comparing cultures with ibuprofen with those without.

Techniques Used: Incubation

FIGURE 7. Expression of SOCS1, Stat1, and SOCS3 in the three MEF strains. mRNA and protein levels of SOCS1, Stat1 (A–C), and SOCS3 (D, E, and G) in WT, IL-18 ko, and IL-18R ko MEF were assayed by real time PCR (A, B, E, and G; differences in gene expression were calculated using the CT method) or Western blotting (C and D). A, B, E, and G, the panels comprise three independent experiments, which are shown as mean changes S.E. 4 h past stimulation. *, p 0.05; **, p 0.01; ***, p 0.001 for both ko strains versus WT. ##, p 0.01; ###, p 0.001 for IL-18 ko versus IL-18R ko. A and E, constitutive levels of the target genes. mRNA quantity in WT MEF is defined as 1; the bars show the changes conferred by the knockouts in percentage, none of which are significant. B and G, the graphs represent induction in SOCS1, Stat1, and SOCS3 mRNA by 25 ng/ml IFN as -fold increase over base lines. F, normalized IL-6 levels in the supernatants from the same IFN-stimulated MEF cultures S.E. are depicted. C and D, MEF cells from the three strains were stimulated with the indicated concentrations of IFN (C) or 25 ng/ml IFN (D) for 20 h. Lysates were then analyzed by Western blotting with staining for SOCS1, SOCS3, or -actin. One blot representative of four others with similar results is shown.

Figure Legend Snippet: FIGURE 7. Expression of SOCS1, Stat1, and SOCS3 in the three MEF strains. mRNA and protein levels of SOCS1, Stat1 (A–C), and SOCS3 (D, E, and G) in WT, IL-18 ko, and IL-18R ko MEF were assayed by real time PCR (A, B, E, and G; differences in gene expression were calculated using the CT method) or Western blotting (C and D). A, B, E, and G, the panels comprise three independent experiments, which are shown as mean changes S.E. 4 h past stimulation. *, p 0.05; **, p 0.01; ***, p 0.001 for both ko strains versus WT. ##, p 0.01; ###, p 0.001 for IL-18 ko versus IL-18R ko. A and E, constitutive levels of the target genes. mRNA quantity in WT MEF is defined as 1; the bars show the changes conferred by the knockouts in percentage, none of which are significant. B and G, the graphs represent induction in SOCS1, Stat1, and SOCS3 mRNA by 25 ng/ml IFN as -fold increase over base lines. F, normalized IL-6 levels in the supernatants from the same IFN-stimulated MEF cultures S.E. are depicted. C and D, MEF cells from the three strains were stimulated with the indicated concentrations of IFN (C) or 25 ng/ml IFN (D) for 20 h. Lysates were then analyzed by Western blotting with staining for SOCS1, SOCS3, or -actin. One blot representative of four others with similar results is shown.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Staining

FIGURE 8. Reduction of IL-18R in human PBMC. PBMC from 10 healthy volunteers were transfected with concentration-matched pairs of 250 nM siRNA to IL-18R or scrambled siRNA. A, cells were stimulated with 1 g/ml LPS or 25 ng/ml IL-12 and 50 ng/ml IL-18. Supernatants were harvested after 20 h, and cytokine levels were determined by multiplex or regular ELISA. Means S.E. of absolute cytokine concentrations from 7 of the 10 donors are shown. *, p 0.05; **, p 0.01; ***, p 0.001 for siIL-18R versus scrambled. B, cells from four donors were lysed 2 and 20 h after stimulation. RNA was isolated and subjected to reverse transcription and quantitative real time PCR. Changes in mRNA expression were calculated using the CT method. Scrambled controls were set at 100% for each condition; percentage changes in IL-18R mRNA levels normalized to -actin and glyceraldehyde-3-phos- phate dehydrogenase are shown. C, lysates from the same experiment were subjected to Western blot analysis with staining for SOCS3. One representa- tive of three blots giving similar results is shown. The bands depicted are from one gel; between the pair, one lane containing a condition not pertaining to this study was cut out.

Figure Legend Snippet: FIGURE 8. Reduction of IL-18R in human PBMC. PBMC from 10 healthy volunteers were transfected with concentration-matched pairs of 250 nM siRNA to IL-18R or scrambled siRNA. A, cells were stimulated with 1 g/ml LPS or 25 ng/ml IL-12 and 50 ng/ml IL-18. Supernatants were harvested after 20 h, and cytokine levels were determined by multiplex or regular ELISA. Means S.E. of absolute cytokine concentrations from 7 of the 10 donors are shown. *, p 0.05; **, p 0.01; ***, p 0.001 for siIL-18R versus scrambled. B, cells from four donors were lysed 2 and 20 h after stimulation. RNA was isolated and subjected to reverse transcription and quantitative real time PCR. Changes in mRNA expression were calculated using the CT method. Scrambled controls were set at 100% for each condition; percentage changes in IL-18R mRNA levels normalized to -actin and glyceraldehyde-3-phos- phate dehydrogenase are shown. C, lysates from the same experiment were subjected to Western blot analysis with staining for SOCS3. One representa- tive of three blots giving similar results is shown. The bands depicted are from one gel; between the pair, one lane containing a condition not pertaining to this study was cut out.

Techniques Used: Transfection, Concentration Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining

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Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE 1. IL-18R in the three MEF strains. MEF cells grown for 2 days were stained against mouse IL-18R chain and visualized using confocal digital microscopy. Red dye, anti-IL-18R; blue dye, nuclei; green dye, cell membranes (the last only shown in insets). A, WT; B, IL-18 ko; C, IL-18R ko. The insets show each strain stained with an IgG control instead of the anti-IL-18R antibody.

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques: Staining, Microscopy, Control

Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE 2. Production of MIP-1 in IL-1- and TNF-stimulated MEF cells. WT, IL-18 ko, and IL-18R ko MEF cells were stimulated with the indicated concentrations of either IL-1 or TNF, and after 20 h (A, B, and D) or 6 h (C; 25 ng/ml IL-1), the levels of MIP-1 were measured in the supernatants. Results represent MIP-1 in pg/mg total protein in IL-1-exposed (A, C, and D) or TNF-exposed (B and D) cells. The data are means S.E. of four independent experiments, each experiment performed on a separate cell line derived from a different mother (see Table 1). A–C, *, p 0.05; ***, p 0.001 compared with the values in WT cells. D, comparison of IL-1 (1 ng/ml) with TNF (10 ng/ml) as stimuli of MEF cells. Data are means S.E. (n 4); *, p 0.05; ***, p 0.001 for IL-1- compared with TNF-stimulated MEF.

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques: Derivative Assay, Comparison

Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE 3. Differential induction of IL-1, IL-6, and PGE2 in the MEF cell strains. WT, IL-18 ko, and IL-18R ko were stimulated with IL-1 or TNF. After 20 h, supernatants were removed and used to generate D and E and the data shown in Fig. 2. The cells were lysed, and levels of IL-1 were measured. The graphs showmeansofnormalizedcytokineorprostaglandinconcentrationsS.E.(n4).A,B,D,andE,*,p0.05;**,p0.01;***,p0.001comparingthekostrains with WT cells. u and v, values in wild type (u) and/or IL-18 ko MEF (v) were below the detection levels of the ECL. A and D, note the breaks in IL-18R bars. E, #, p 0.05; ##, p 0.01 for IL-18 ko versus IL-18R ko. C, 1 ng/ml IL-1 versus 10 ng/ml TNF. The data are mean S.E. of the same four independent experiments as in Fig. 2D. *, p 0.05 for IL-1-stimulated compared with TNF-stimulated cells.

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE 4. Effect of an anti-IL-18R blocking antibody on cytokine production. Increasing concentrations of an anti-IL-18R monoclonal blocking antibody (shown below the horizontal axis in ng/ml) were added to WT or IL-18 ko MEF cells. After 20 h of incubation, the levels of spontaneous IL-6 were measured in the supernatant. A, the level of IL-6 in the absence of anti-IL-18R was set at 1.0 for each of three independent experiments, and the -fold increase was calculated. The data are mean S.E. -fold increase. *, p 0.05; **, p 0.01; ***, p 0.001 compared with in the absence of the antibody. B, an experiment similar to A, except IL-1 (1 ng/ml) was added to the cells 20 min after the anti-IL-18R antibody. The data in C and D are from the same cultures as shown in A and B. In C and D, MIP-1 was measured in the supernatants. **, p 0.01; ***, p 0.001 compared with no antibody.

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques: Blocking Assay, Incubation

Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE 5. Silencing of the IL-18R in A549 lung epithelial cells. Using shRNA, stable A549 cell lines depleted of the IL-18R -chain were created. For control, A549 cells were transfected with scrambled shRNA. The knockdown of the IL-18R chain was assessed by Western blotting (A) and real time PCR (B). A, the arrows indicate the clones used in the experiments. C, the three scrambled-transfected and two shIL-18R-transfected cell lines shown in A and B were stimulated with 10 ng/ml IL-1 or were left untreated for 20 h. Thereafter, IL-6 levels were determined. The panel depicts means of normal- ized cytokine concentrations S.E.; n 6 clones times 4 independent exper- iments; ***, p 0.001 for shIL-18R versus small hairpin scrambled (shscrambled).

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques: shRNA, Control, Transfection, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Clone Assay

Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE6.EffectsofibuprofenandIL-1RaontheproductionofIL-6inMEF cells. A, constitutive secretion of IL-6 was measured in the absence or pres- ence of the IL-1Ra. IL-1Ra was added to the cultures of IL-18R ko cells at 10 g/ml, and after 20 h, levels of IL-6 were measured. In cultures without IL-1Ra (), the level of IL-6 was set at 100%, and the mean percentage change of parallel cultures with IL-1Ra was calculated. Data are mean S.E. percentage change from three independent experiments. ***, p 0.001 compared with cultures without IL-1Ra. B, constitutive production of IL-6 in WT, IL-18 ko, and IL-18R ko MEF cells in the absence and presence of ibuprofen (10 M) after 20 h of incubation. C, each of the MEF strains were stimulated with IL-1 (1 ng/ml) in the absence or presence of ibuprofen (ibu; 10 M). After 20 h, levels of IL-6 were measured in the supernatants. Note the break in the IL-18R bar. B and C,thedataaremeanS.E.ofthreeindependentexperiments.*,p0.05;**,p 0.01; ***, p 0.001 comparing cultures with ibuprofen with those without.

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques: Incubation

Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE 7. Expression of SOCS1, Stat1, and SOCS3 in the three MEF strains. mRNA and protein levels of SOCS1, Stat1 (A–C), and SOCS3 (D, E, and G) in WT, IL-18 ko, and IL-18R ko MEF were assayed by real time PCR (A, B, E, and G; differences in gene expression were calculated using the CT method) or Western blotting (C and D). A, B, E, and G, the panels comprise three independent experiments, which are shown as mean changes S.E. 4 h past stimulation. *, p 0.05; **, p 0.01; ***, p 0.001 for both ko strains versus WT. ##, p 0.01; ###, p 0.001 for IL-18 ko versus IL-18R ko. A and E, constitutive levels of the target genes. mRNA quantity in WT MEF is defined as 1; the bars show the changes conferred by the knockouts in percentage, none of which are significant. B and G, the graphs represent induction in SOCS1, Stat1, and SOCS3 mRNA by 25 ng/ml IFN as -fold increase over base lines. F, normalized IL-6 levels in the supernatants from the same IFN-stimulated MEF cultures S.E. are depicted. C and D, MEF cells from the three strains were stimulated with the indicated concentrations of IFN (C) or 25 ng/ml IFN (D) for 20 h. Lysates were then analyzed by Western blotting with staining for SOCS1, SOCS3, or -actin. One blot representative of four others with similar results is shown.

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Staining

Journal: Journal of Biological Chemistry

Article Title: Increased Cytokine Production in Interleukin-18 Receptor α-deficient Cells Is Associated with Dysregulation of Suppressors of Cytokine Signaling

doi: 10.1074/jbc.m109.004184

Figure Lengend Snippet: FIGURE 8. Reduction of IL-18R in human PBMC. PBMC from 10 healthy volunteers were transfected with concentration-matched pairs of 250 nM siRNA to IL-18R or scrambled siRNA. A, cells were stimulated with 1 g/ml LPS or 25 ng/ml IL-12 and 50 ng/ml IL-18. Supernatants were harvested after 20 h, and cytokine levels were determined by multiplex or regular ELISA. Means S.E. of absolute cytokine concentrations from 7 of the 10 donors are shown. *, p 0.05; **, p 0.01; ***, p 0.001 for siIL-18R versus scrambled. B, cells from four donors were lysed 2 and 20 h after stimulation. RNA was isolated and subjected to reverse transcription and quantitative real time PCR. Changes in mRNA expression were calculated using the CT method. Scrambled controls were set at 100% for each condition; percentage changes in IL-18R mRNA levels normalized to -actin and glyceraldehyde-3-phos- phate dehydrogenase are shown. C, lysates from the same experiment were subjected to Western blot analysis with staining for SOCS3. One representa- tive of three blots giving similar results is shown. The bands depicted are from one gel; between the pair, one lane containing a condition not pertaining to this study was cut out.

Article Snippet: Staining was performed overnight at 4 °C using a biotinylated goat anti-mouse IL-18R antibody (2.5 g/ml; R&D Systems) in PBS containing 1% BSA.

Techniques: Transfection, Concentration Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Staining

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