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

Article Title: The epistatic interrelationships of IL-1, IL-1 receptor antagonist, and the type I IL-1 receptor.

doi: 10.4049/jimmunol.169.1.393

Figure Lengend Snippet: FIGURE 1. Murine protein isoforms of IL-1ra. A, Schematic diagram showing the relationships between the murine IL-1ra protein isoforms. Shared sequences are in black, those unique to pro-sIL-1ra (namely the signal peptide) are in gray, those unique to icIL-1ra1 (alternate exon 1) are in white, and the N-terminus that is deleted from icIL-1ra3 is hatched. B and C, Detection of the various proteins isoforms by Western blot of liver proteins from mice challenged with LPS (B) and skin proteins from un- challenged mice (C) of wt, rako, ratg, and rako, ratg mutants. The different isoforms of IL-1ra were assigned based on size relative to m.w. markers and recombinant sIL-1ra (R&D Systems), partial sequencing, and tissue distribution.

Article Snippet: ELISAs were performed as previously described and followed the manufacturers’ suggested protocols using the following Abs or kits: polyclonal goat anti-mIL-1ra (R&D Systems, Minneapolis, MN); DuoSet kits for IL-1 , IL-10, and TNF- (R&D Systems); and OptEIA for IFN- (BD PharMingen, San Diego, CA).

Techniques: Western Blot, Recombinant, Sequencing

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

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 1. Generation of icIL-1Ra12/2 mice. (A) Targeting strategy used for the introduction of an EGFP–Neo cassette into the icIL-1Ra1–specific first exon (icIl1rn1) of the mouse Il1rn gene. This approach does not interfere with the expression of the other transcript variants. Shown from top to bottom are the WT icIl1rn1 genomic locus (WT allele), the targeting vector and the targeted genomic locus (Mutant allele). Bold lines indicate arms of homology for recombination. Exon 1 is represented as a gray box and numbered. The neomycin resistance gene (Neor) is flanked by two loxP sites (triangles). The primers used to identify recombinant ES cells are indicated as S1 and S2. (B) Southern blot analysis of WT (+/+) and heterozygous (+/2) (one WT allele, one mutant allele) ES cells using a 59 external probe (left panel) or a 39 internal probe (right panel). The WT (6.4-kb [BglII digestion] or 8.9-kb [HindIII digestion] bands) and mutant alleles (7.4-kb [BglII digestion] or 6.2-kb [HindIII digestion] bands) were detected in BglII- or HindIII-digested ES cell genomic DNA, using, respectively, the 59 or 39 probes shown in (A). (C) PCR genotyping to distinguish WT (+/+), heterozygous (+/2) and icIL-1Ra1(2/2) mice. The primers used are indicated as P1, P2, and P3 in (A). The sizes of the WT and icIL-1Ra12/2 PCR products are 300 and 842 bp, respectively. (D) Male (left panel) and female (right panel) WT (male: n = 13/n = 6 [10/17 wk]; female: n = 11/n = 6 [10/17 wk]) and icIL-1Ra12/2 (male: n = 13/n = 13 [10/17 wk]; female: n = 11/n = 9 [10/17 wk]) mice were weighed at 10 and 17 wk of age. Data are shown as the mean 6 SEM. No significant differences in body weight were observed between the genotypes (unpaired two-tailed Student t test). (E) Analysis of IL-1Ra isoforms in lysates of BMDC generated from naive WT and icIL-1Ra12/2 (KO) mice. IL-1Ra was detected using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression. One representative sample per genotype out of n = 3 per genotype is shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms are as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa (13, 69).

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Recombinant, Southern Blot, Two Tailed Test, Generated

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

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 2. Characterization of BMDC and BMDM from WT and icIL-1Ra12/2 mice. (A) Determination of mRNA levels for icIL-1Ra1 (icIl1rn1) and for sIL-1Ra and icIL-1Ra3 (sIl1rn) in BMDC (left panel) and BMDM (right panel). BMDC and BMDM from naive WT (n = 5) and icIL-1Ra12/2 (n = 5) mice were left untreated (control) or stimulated with 100 ng/ml LPS for 24 h. Total RNA was isolated for qRT-PCR analysis. Results represent icIl1rn1 and sIl1rn mRNA expression levels relative to Mrpl32 mRNA levels. (B) Release of IL-1Ra protein (all isoforms) into the supernatant. BMDC (left panel) and BMDM (right panel) from naive WT (n = 5) and icIL-1Ra12/2 (n = 5) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra in the supernatant was measured using an ELISA that recognizes all isoforms of IL-1Ra. (A and B) Data are shown as the mean 6 SEM of values obtained from three (BMDC) or two (BMDM) independent experiments. The unpaired or paired two-tailed Student t test was used to test statistical significance. All p values ,0.05 were considered to be significant. (C) Analysis of IL-1Ra isoforms in lysates and cell supernatants of BMDC (left panel) and BMDM (right panel) by Western blot. BMDC and BMDM from naive WT and icIL-1Ra12/2 (KO) mice were left untreated (control) or stimulated with LPS for 24 h. IL-1Ra isoforms were analyzed in cell lysates or collected supernatants (30 ml) using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra isoforms. Protein loading was assessed by the determination of GAPDH expression in lysates. One nanogram of rsIL-1Ra served as control. Two rep- resentative samples out of n = 5 per genotype are shown. Arrows indicate different IL-1Ra isoforms. Theoretical molecular mass of IL-1Ra isoforms is as follows: prosIL-1Ra1, 20 kDa; mature sIL-1Ra1, between 17 and 22 kDa; icIL-1Ra1, 18 kDa; icIL-1Ra3, 16 kDa. n.d., not detected. SN, supernatant.

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Control, Isolation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot

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

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 3. icIL-1Ra1 deficiency in- creases the clinical severity of Aldara- induced ear thickness and the expression of proinflammatory cytokines. (A) IL- 1Ra isoforms were analyzed in lysates of total dorsal skin from naive WT, hetero- zygous (HET) and icIL-1Ra12/2 (KO) mice using a polyclonal goat anti-mouse IL-1Ra Ab recognizing all IL-1Ra iso- forms. Protein loading was assessed by the determination of GAPDH expression. One representative sample out of n = 3 per genotype is shown. Arrow indicates the icIL-1Ra1 isoform. Theoretical mo- lecular mass of icIL-1Ra1: 18 kDa. (B–E) Aldara, or Vaseline as a control, were applied on the right or left ear, respec- tively, of WT (n = 21) and icIL-1Ra12/2

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Expressing, Control

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

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 4. icIL-1Ra1 is the main IL-1Ra isoform regulating Aldara-induced skin inflammation. (A) Representative immunohistochemical detection of IL-1Ra (brown staining) on ear sections of WT and icIL-1Ra12/2 mice on day 8 after daily application of Vaseline (control) or Aldara. Scale bar, 100 mm. (B) Analysis of IL-1Ra isoforms in lysate of Vaseline-treated (control) (upper panel) and Aldara-treated (lower panel) ears from WT and icIL-1Ra12/2

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Immunohistochemical staining, Staining, Control

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

Article Title: Intracellular IL-1 Receptor Antagonist Isoform 1 Released from Keratinocytes upon Cell Death Acts as an Inhibitor for the Alarmin IL-1α.

doi: 10.4049/jimmunol.1901074

Figure Lengend Snippet: FIGURE 5. icIL-1Ra1 plays no intracellular role in keratinocytes but is released together with IL-1a upon Aldara treatment. (A) Differential gene expression analysis based on RNA-Seq data of primary keratinocytes from icIL-1Ra12/2 versus WT mice. Primary keratinocytes were isolated from WT (n = 4) and icIL-1Ra12/2 (n = 4) mouse tails and left untreated as a control (left panel) or stimulated with 100 ng/ml rec. m IL-1a (middle panel) or 100 ng/ml rec. m IL-36b (right panel) for 6 h. Total RNA was then isolated from keratinocytes for RNA-Seq analysis. A total of 10,750, 10,709, or 10,727 genes were tested for control, IL-1a–, or IL-36b–treated cells, respectively. The fold-change threshold and Benjamini–Hochberg corrected p value threshold were set to 2 and 0.05, respectively. (B) Ear explants taken from WT (n = 6) and icIL-1Ra12/2 (n = 6) mice at day 8 after daily treatment with Vaseline (control) or Aldara were cultured for 24 h. Levels of IL-1Ra, IL-1a, and IL-1b in the supernatant or the percentage of cytotoxicity were assessed by ELISA or LDH assay, respectively. The IL-1Ra ELISA recognizes all isoforms of IL-1Ra. Data are shown as the mean 6 SEM from one (Figure legend continues)

Article Snippet: In brief, tissue sections were incubated with 7.2 mg/ml polyclonal goat anti-mouse IL-1Ra Ab (capture Ab of the mouse IL-1Ra DuoSet ELISA Kit; R&D Systems, Minneapolis, MN) overnight at 4 ̊C.

Techniques: Gene Expression, RNA Sequencing, Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal:

Article Title: Genetic alterations of IL-1 receptor antagonist in mice affect plasma cholesterol level and foam cell lesion size

doi: 10.1073/pnas.092324399

Figure Lengend Snippet: IL-1ra deficiency is associated with decreased non-HDL plasma cholesterol and increased foam cell lesion size. Wild-type (n = 24), IL-1ra ± (Heterozyg. IL-1ra KO; n = 37), and IL-1ra −/− (Homozyg. IL-1ra KO; n = 19) mice on the C57BL/6J background were fed a cholate-containing cholesterol-enriched diet for 3 mo and then analyzed for total plasma and HDL cholesterol concentrations (A) and foam cell lesion area in the proximal aorta (B). (A) The homozygous IL-1ra knockout total plasma cholesterol value was statistically different from the wild-type knockout values (P < 0.05). (B) The square root-transformed values for wild-type, heterozygous knockout, and homozygous knockout lesion sizes were 59.9 ± 5.8, 71.9 ± 5.2, and 77.2 ± 17.3 μm, respectively (P > 0.05). For an explanation of the statistical analysis of these data, see Statistical Analysis.

Article Snippet: Next, the sections were incubated with 1.5% donkey serum containing 2 ng/μl of goat polyclonal anti-IL-1ra N-terminal peptide IgG (Q-19 from Santa Cruz Biotechnology) for 16 h at 4°C.

Techniques: Knock-Out, Transformation Assay

Journal:

Article Title: Genetic alterations of IL-1 receptor antagonist in mice affect plasma cholesterol level and foam cell lesion size

doi: 10.1073/pnas.092324399

Figure Lengend Snippet: IL-1ra protein is present in the endothelium of the proximal aorta of cholate/cholesterol-fed C57BL/6J mice. C57BL/6J wild-type (WT; A) and IL-1ra −/− (IL-1ra KO; B) mice were fed the cholate/cholesterol diet for 7 wk. Sections of proximal aorta from these mice were then subjected to immunohistochemical analysis by using an anti-IL-1ra Ab. (Insets) Enlargements of the sections outlined by the dotted box in each A and B.

Article Snippet: Next, the sections were incubated with 1.5% donkey serum containing 2 ng/μl of goat polyclonal anti-IL-1ra N-terminal peptide IgG (Q-19 from Santa Cruz Biotechnology) for 16 h at 4°C.

Techniques: Immunohistochemical staining

Journal:

Article Title: Genetic alterations of IL-1 receptor antagonist in mice affect plasma cholesterol level and foam cell lesion size

doi: 10.1073/pnas.092324399

Figure Lengend Snippet: IL-1ra over-expression is associated with increased non-HDL plasma cholesterol in LDLR knockout mice fed a cholesterol-fat diet. LDLR knockout mice (LDLR KO/No Tg; n = 31) and LDLR knockout mice homozygous for the IL-1ra transgene (LDLR KO/IL-1ra Tg; n = 18) were fed a cholesterol-fat enriched “Western” diet for 10 wk and then analyzed for total plasma cholesterol concentrations (A), plasma cholesterol FPLC profile (B), and lesion area in the proximal aorta (C). (A) IL-1ra transgenic total plasma cholesterol and triglyceride values were statistically different from the wild-type values (P = 0.001 and 0.003, respectively). (C) Square root-transformed values for wild-type and transgenic lesion sizes were 455.8 ± 17.6 and 450.8 ± 23.1 μm, respectively (P > 0.05).

Article Snippet: Next, the sections were incubated with 1.5% donkey serum containing 2 ng/μl of goat polyclonal anti-IL-1ra N-terminal peptide IgG (Q-19 from Santa Cruz Biotechnology) for 16 h at 4°C.

Techniques: Over Expression, Knock-Out, Western Blot, Transgenic Assay, Transformation Assay

Journal:

Article Title: Genetic alterations of IL-1 receptor antagonist in mice affect plasma cholesterol level and foam cell lesion size

doi: 10.1073/pnas.092324399

Figure Lengend Snippet: IL-1ra over-expression is associated with decreased foam cell lesion size in LDLR knockout mice fed a cholate/cholesterol diet. LDLR knockout mice (LDLR KO/No Tg; n = 36) and LDLR knockout mice homozygous for the IL-1ra transgene (LDLR KO/IL-1ra Tg; n = 39) were fed a cholate-containing cholesterol-enriched diet for 4 wk and then analyzed for total plasma cholesterol concentrations (A), plasma cholesterol FPLC profile (B), and lesion area in the proximal aorta (C). In A, the IL-1ra transgenic total plasma cholesterol value was statistically different from the wild-type value (P = 0.01). In C, the square root-transformed values for wild-type and transgenic lesion sizes were 222.7 ± 8.8 and ± 199.4 ± 7.9 μm, respectively (P = 0.027).

Article Snippet: Next, the sections were incubated with 1.5% donkey serum containing 2 ng/μl of goat polyclonal anti-IL-1ra N-terminal peptide IgG (Q-19 from Santa Cruz Biotechnology) for 16 h at 4°C.

Techniques: Over Expression, Knock-Out, Transgenic Assay, Transformation Assay

Journal:

Article Title: Genetic alterations of IL-1 receptor antagonist in mice affect plasma cholesterol level and foam cell lesion size

doi: 10.1073/pnas.092324399

Figure Lengend Snippet: IL-1ra protein is present in increased amounts in the proximal aorta of IL-1ra transgenic LDLR knockout mice. LDLR knockout mice homozygous for the IL-1ra transgene (IL-1ra Tg; A and C) and LDLR knockout mice without the transgene (no Tg; B and D) were fed the cholate/cholesterol diet for 4 wk. Sections of proximal aorta from these mice were then subjected to immunohistochemical analysis by using an Ab directed against IL-1ra. The sections in C and D were probed with the anti-IL-1ra Ab that had been preabsorbed with the IL-1ra peptide antigen (Preabs Ab). Note that staining above background is present in the section from the nontransgenic mouse (B), but the staining is much more intense in the section from the transgenic mouse (A).

Article Snippet: Next, the sections were incubated with 1.5% donkey serum containing 2 ng/μl of goat polyclonal anti-IL-1ra N-terminal peptide IgG (Q-19 from Santa Cruz Biotechnology) for 16 h at 4°C.

Techniques: Transgenic Assay, Knock-Out, Immunohistochemical staining, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Reprogramming of a subpopulation of human blood neutrophils by prolonged exposure to cytokines.

doi: 10.1038/labinvest.2009.74

Figure Lengend Snippet: Figure 8 Release of IL-1b, IL-8 and IL-1Ra by long-lived neutrophils. Freshly isolated neutrophils (bar 1) and long-lived neutrophils (bar 2) were incubated at 5 106/ml for 24 h. IL-1b (c), IL-1Ra (b) and IL-8 (a) were measured in supernatants using enzyme immunometric assays. The results are means±s.e.m. of three donors (freshly isolated neutrophils) and five donors (long-lived neutrophils). Statistics: Student’s unpaired t-test for freshly isolated neutrophils vs long-lived neutrophils (*Po0.05, ***Po0.001).

Article Snippet: Apostat intracellular caspase detection kit (FITC-VD-FMK), EIA kit for IL-1Ra (biotinylated secondary goat anti-human IL-1Ra Ab: DY280, #1144097) and Proteome Profiler: Human Phospho-MAP Kinase Array Kit were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Incubation

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced Evans blue extravasation and BBB ultrastructure. a Experimental protocol. b , c Extravasation of Evans blue dye in brain tissues of the control, vehicle, IL-1RA, and IL-1RA-PEP groups was evaluated quantitatively. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 6 per group, based on one-way ANOVA with Bonferroni correction. d Ultrastructure changes in the BBB. The basement membranes (marked between black arrows in top panels) were observed in different groups. The black frames in middle panels are areas selected for amplification in the following bottom panels, to highlight tight junctions (pointed by white arrows). Scale bar, 500 nm and 2 μm for the inserts. MCAO middle cerebral artery occlusion, BBB blood-brain barrier

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Control, Amplification

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced endogenous IgG leakage and permeability of FITC-dextran. a Extravasation of endogenous IgG, and intravenous injection and measurement of FITC-dextran tracer were evaluated in the different groups. Increases (%) in IgG ( b ) and FITC-dextran ( c ) were represented by the proportion of the areas of positive regions in the cerebral cortex of the ipsilateral hemisphere. Scale bar, 100 and 200 μm for the inserts. Data are shown as mean ± SEM; ** P < 0.01, *** P < 0.001 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 6 per group, based on one-way ANOVA with Bonferroni correction. MCAO middle cerebral artery occlusion, FITC-dextran fluorescein isothiocyanate–dextran

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Permeability, Injection

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Brain penetration effects and levels of IL-1RA and IL-1RA-PEP. a , b At 24 h after ischemia-reperfusion, the permeation effect on brain tissue was assessed by immunohistochemical staining. The rats with middle cerebral artery occlusion (MCAO) in separate groups were injected intravenously with IL-1RA or IL-1RA-PEP. c , d High magnification of double immunofluorescence images show co-localization of IL-1RA with NeuN (arrows) and IL-1RA-PEP with NeuN (arrows). Scale bar, 5.0 mm and 50 μm for the inserts. e The permeation effect was expressed as a percentage increase in staining intensity in the cerebral cortex and striatum of the contralateral hemisphere. f The concentration of IL-1RA in the ipsilateral ischemic brain hemisphere of rats subjected to MCAO, 4 h after a single intravenous injection of IL-1RA (50 mg/kg) or IL-1RA-PEP (50 mg/kg), was measured by ELISA. Panels show IL-1RA concentration in the cerebral cortex and striatum area, respectively. Data are shown as mean ± SEM; * P < 0.05 in comparison with the rat intravenously injected with IL-1RA, n = 4–6 per group, based on the Student’s t test with Welch’s correction

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Immunohistochemical staining, Staining, Injection, Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced changes in tight junction protein expression and localization. a–e Protein expression levels of ZO-1, occludin, and claudin-5 in cerebral tissue of the control, vehicle, IL-1RA, and IL-1RA-PEP groups were evaluated. f–i Localization of ZO-1, occludin, and claudin-5 in the cerebral tissue of different groups was also analyzed. Scale bar = 50 μm. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 4 per group, based on one-way ANOVA with Bonferroni correction. MCAO middle cerebral artery occlusion

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on transient MCAO-induced changes in MMP protein expression and localization. a–c Protein expression levels of MMP-9 and MMP-2 in cerebral tissue of the control, vehicle, IL-1RA, and IL-1RA-PEP groups were evaluated. d–f Localization of MMP-9 and MMP-2 in the cerebral tissue of different groups was also analyzed. Scale bar = 100 μm for the inserts. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group, # P < 0.05 compared to the IL-1RA group, n = 4 per group, based on one-way ANOVA with Bonferroni correction. MCAO middle cerebral artery occlusion

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Expressing, Control

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Effects of IL-1RA and IL-1RA-PEP on angiopoietin-1 and VEGF expression in brain tissues following ischemia-reperfusion. a–c Protein expression levels of angiopoietin-1 and vascular endothelial growth factor (VEGF) were analyzed by western blot. The gray value of the band was scanned by optical densitometry, and the ratio value was statistically analyzed. d , e The mRNA expression of angiopoietin-1 and VEGF were analyzed by real-time quantitative PCR. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group, n = 6 per group, based on one-way ANOVA with Bonferroni correction

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

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

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Role of p65/NF-κB pathways in IL-1RA-PEP-mediated effects on blood-brain barrier (BBB) disruption. a After the JSH-23 inhibitor was administered, expression levels of ZO-1, MMP-9, angiopoietin-1, and VEGF were analyzed by western blot in the different groups. b–e The gray value of the band was scanned by optical densitometry, and the ratio value was statistically analyzed. Data are shown as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the vehicle group without JSH-23; # P < 0.05, ## P < 0.01 compared to the IL-1RA-PEP group without JSH-23; n = 4 per group, based on one-way ANOVA with Bonferroni correction

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Disruption, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: A novel IL-1RA-PEP fusion protein alleviates blood-brain barrier disruption after ischemia-reperfusion in male rats

doi: 10.1186/s12974-018-1058-z

Figure Lengend Snippet: Graphic summary for the effect of IL-1RA and IL-1RA-PEP on BBB disruption in rats subjected to cerebral ischemia-reperfusion injury

Article Snippet: They were then blocked with 10% normal rabbit serum for 30 min and incubated overnight at 4 °C with goat anti-human IL-1RA antibody (1:500, R&D Systems, Inc., Minneapolis, USA) and rabbit anti-NeuN antibody to label neuronal cells (1:100, Chemicon, Hampshire, UK).

Techniques: Disruption