Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36α was induced in UUO kidney and renal TECs. (A) Real-time PCR showing relative renal levels of IL-36 mRNAs in sham control and UUO mice. (B) IHC staining showing IL-36α protein expression in sham control and UUO mice; the arrows indicate IL-36α–positive renal TECs of atrophic tubules. Original magnification, ×400. (C) Quantitative analysis for the IL-36α–positive tubules. Data are means±SEM of nine mice per group. Western blot analysis for IL-36α protein expression in mouse tubular epithelial cells (mTECs) M-1 under (D) mechanically induced pressure, (E) H2O2, or (F) HMGB-1. (G) Semiquantitative IL-36α levels in mTECs M-1 lysates. Western blot analysis for IL-36α protein expression in house tubular epithelial cells (hTECs) HK-2 under (H) mechanically induced pressure, (I) H2O2, or (J) HMGB-1. (K) Semiquantitative IL-36α levels in hTECs HK-2 lysates. Data are means±SEM of three replicative results obtained from in vitro experiments. *P<0.05; **P<0.01; ***P<0.01; #not detectable.

Article Snippet: 50 The target proteins were probed with respective primary antibodies against mouse IL-36 α (AF2297; R&D), β -actin (sc-130656; Santa Cruz Biotech, Santa Cruz, CA), human IL-36 α (AP5234c; Abgent), NLRP3 (AG-20B-0014; Adipogen, San Diego, CA), IL-18 (sc-7954; Santa Cruz Biotech), caspase-1 (AG-20B-0044; Adipogen), or IL-1 β (AF-401; R&D) followed by the addition of HRP-conjugated secondary antibodies (Santa Cruz Biotech).

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

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling facilitated NLRP3 inflammasome activation in renal tissues of UUO mice and renal TECs. (A) Representative Western blots analysis and semiquantitation for NLRP3, IL-18, and IL-1β in renal tissues. (B) Caspase-1 activity determined by an activity assay in renal tissues. (C) ELISA for urinary protein levels of IL-1β in normal (sham control) or pelvic (UUO) urine samples. Data are means±SEM of nine mice per group. M-1 TECs treated with (D) rIL-36α, LPS (2 μg/ml), or saline for 24 hours (representative Western blots and semiquantitation for NLRP3, pro–IL-18, and pro–IL-1β in lysates) or (E) rIL-36α, LPS, or saline for 24 hours and an additional 1 hour of ATP incubation (representative Western blots for IL-18, IL-1β, and caspase-1 p10 in supernatants). (F) Caspase-1 activity determined by an activity assay in lysate. Data are means±SEM of three replicative results obtained from in vitro experiments. *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: 50 The target proteins were probed with respective primary antibodies against mouse IL-36 α (AF2297; R&D), β -actin (sc-130656; Santa Cruz Biotech, Santa Cruz, CA), human IL-36 α (AP5234c; Abgent), NLRP3 (AG-20B-0014; Adipogen, San Diego, CA), IL-18 (sc-7954; Santa Cruz Biotech), caspase-1 (AG-20B-0044; Adipogen), or IL-1 β (AF-401; R&D) followed by the addition of HRP-conjugated secondary antibodies (Santa Cruz Biotech).

Techniques: Activation Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation, In Vitro

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling facilitated NLRP3 inflammasome activation in cultured macrophages. (A) ELISA analysis of IL-1β protein levels in supernatant of murine J774A.1 macrophages incubated for 30 minutes with or without rIL-36α (150 ng/ml), 5.5 hours with or without 0.5 μg/ml LPS, and 30 minutes with or without 5 mM ATP. (B) Representative Western blots and semiquantitation for NLRP3 and pro–IL-1β in lysates of J774A.1 macrophages incubated with or without rIL-36α (150 ng/ml) for 30 minutes and then treated with or without LPS for 6 hours. (C) Representative Western blots for caspase-1 p10 in supernatants of J774A.1 incubated with or without rIL-36α (150 ng/ml), 5.5 hours with or without 0.5 μg g/ml LPS, and 30 minutes with or without 5 mM ATP. (D) ELISA analysis of IL-1β protein levels in supernatant of peritoneal macrophages from IL-36R KO and WT mice incubated for 5.5 hours with or without 1 μg g/ml LPS and 30 minutes with or without 5 mM ATP. (E) ELISA analysis of IL-1β protein levels in supernatant of BMDMs from WT, TLR4 KO, and MyD88 KO mice treated with similar conditions as J774A.1 above. (F) Representative Western blots and semiquantitation for NLRP3 and pro–IL-1β in lysates of BMDMs from WT, TLR4 KO, and MyD88 KO mice treated with or without rIL-36α (150 ng/ml). Data are means±SEM of three replicative results obtained from the experiments. Mϕ, macrophage *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: 50 The target proteins were probed with respective primary antibodies against mouse IL-36 α (AF2297; R&D), β -actin (sc-130656; Santa Cruz Biotech, Santa Cruz, CA), human IL-36 α (AP5234c; Abgent), NLRP3 (AG-20B-0014; Adipogen, San Diego, CA), IL-18 (sc-7954; Santa Cruz Biotech), caspase-1 (AG-20B-0044; Adipogen), or IL-1 β (AF-401; R&D) followed by the addition of HRP-conjugated secondary antibodies (Santa Cruz Biotech).

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling activated NLRP3 inflammasome activation in BMDCs. (A) ELISA analysis of IL-1β protein levels in supernatant of BMDCs from WT, IL-36R KO, and NLRP3 KO mice incubated with or without rIL-36α (150 ng/ml) for 24 hours. (B) Representative Western blots and (C) semiquantitation for NLRP3, pro–IL-1β, and caspase-1 p10 in lysates of BMDCs from WT, IL-36R KO, and NLRP3 KO mice incubated with or without rIL-36α (150 ng/ml) for 24 hours. Data are means±SEM of three replicative results obtained from the experiments. *P<0.01; ***P<0.01; #not detectable.

Article Snippet: 50 The target proteins were probed with respective primary antibodies against mouse IL-36 α (AF2297; R&D), β -actin (sc-130656; Santa Cruz Biotech, Santa Cruz, CA), human IL-36 α (AP5234c; Abgent), NLRP3 (AG-20B-0014; Adipogen, San Diego, CA), IL-18 (sc-7954; Santa Cruz Biotech), caspase-1 (AG-20B-0044; Adipogen), or IL-1 β (AF-401; R&D) followed by the addition of HRP-conjugated secondary antibodies (Santa Cruz Biotech).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling facilitated DC-induced T cells activation in UUO mice and cell models. (A) Flow cytometric analysis showing the percentages of CD4+/CD69+ and CD8+/CD69+ in renal draining lymphoid cells at day 7 after UUO or sham operation. Data are means±SEM of nine mice per group. (B) BMDCs were incubated with or without rIL-36α (150 ng/ml) for 16 hours, pulsed with OVA257–264 or OVA323–339 peptide, and then, cocultured with OT-I CD8 or OT-II CD4 T cells, respectively. After 3 days, the T cell proliferation was measured by [3H]thymidine incorporation. Data are means±SEM of three replicative results obtained from the experiments. (C) Flow cytometric analysis showing expression levels CD40, CD80, and CD86 (within gated CD11c cells) in BMDCs from WT and IL-36R KO mice, which were incubated with or without rIL-36α (150 ng/ml) for 24 hours. Data are means±SEM of three replicative results obtained from the experiments. (D) Flow cytometric analysis showing the numbers of CD11c+ cells in renal draining lymphoid cells at day 7 after UUO or sham operation; data are means±SEM of five mice per group. (E) Flow cytometric analysis showing the CD86+CD11c+ cells in renal draining lymphoid cells at day 7 after UUO or sham operation. Data are means±SEM of five mice per group. rdLN, renal draining lymph node. *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: 50 The target proteins were probed with respective primary antibodies against mouse IL-36 α (AF2297; R&D), β -actin (sc-130656; Santa Cruz Biotech, Santa Cruz, CA), human IL-36 α (AP5234c; Abgent), NLRP3 (AG-20B-0014; Adipogen, San Diego, CA), IL-18 (sc-7954; Santa Cruz Biotech), caspase-1 (AG-20B-0044; Adipogen), or IL-1 β (AF-401; R&D) followed by the addition of HRP-conjugated secondary antibodies (Santa Cruz Biotech).

Techniques: Activation Assay, Incubation, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 facilitated Th17 differentiation in UUO mice and in vitro T cell activation. (A) Real-time PCR showing relative renal mRNA levels of IL-23p19 and IL-17A in sham control and day14 UUO mice. Data are means±SEM of nine mice per group. (B) BMDCs were incubated with or without rIL-36α (150 ng/ml) for 16 hours, pulsed with OVA323–339 peptides, and then, cocultured with OT-II CD4 T cells. After 4 days, supernatants were collected, and the production of IL-17A was detected by ELISA. Data are means±SEM of three replicative results obtained from the experiments. (C) Real-time PCR showing mRNA levels of specific transcription factors for different Th cells in OT-II CD4 cocultured with BMDCs (preincubated with or without rIL-36α for 16 hours and then, pulsed with OVA323–339 peptides) for 4 days. Data are means±SEM of three replicative results obtained from the experiments. *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: 50 The target proteins were probed with respective primary antibodies against mouse IL-36 α (AF2297; R&D), β -actin (sc-130656; Santa Cruz Biotech, Santa Cruz, CA), human IL-36 α (AP5234c; Abgent), NLRP3 (AG-20B-0014; Adipogen, San Diego, CA), IL-18 (sc-7954; Santa Cruz Biotech), caspase-1 (AG-20B-0044; Adipogen), or IL-1 β (AF-401; R&D) followed by the addition of HRP-conjugated secondary antibodies (Santa Cruz Biotech).

Techniques: In Vitro, Activation Assay, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: Proposed mechanistic pathways underlying the pathogenic role of IL-36 in the development of renal TILs. IL-36 may be contributed to the pathogenesis of renal TILs, involving the activation of NLRP3 inflammasome and IL-23/IL-17 axis.

Article Snippet: 50 The target proteins were probed with respective primary antibodies against mouse IL-36 α (AF2297; R&D), β -actin (sc-130656; Santa Cruz Biotech, Santa Cruz, CA), human IL-36 α (AP5234c; Abgent), NLRP3 (AG-20B-0014; Adipogen, San Diego, CA), IL-18 (sc-7954; Santa Cruz Biotech), caspase-1 (AG-20B-0044; Adipogen), or IL-1 β (AF-401; R&D) followed by the addition of HRP-conjugated secondary antibodies (Santa Cruz Biotech).

Techniques: Activation Assay

Journal: The Journal of allergy and clinical immunology

Article Title: IL-1 and IL-36 are dominant cytokines in generalized pustular psoriasis

doi: 10.1016/j.jaci.2016.08.056

Figure Lengend Snippet: IL-36α was not expressed by healthy skin (a), found in the cytoplasm of the uppermost layers of viable KC in PV lesions (b) and expressed at a higher intensity by a wider band of KC in GPP lesions (c). IL-36α expression was strongest in the LC proximal to neutrophilic pustules, but not the neutrophils themselves (d). Faint expression of IL-36γ could be detected in the cytoplasm, but was most apparent in the nuclei of healthy skin KC (e). IL-36γ was intensified in PV lesions (f) yet this was more intense in GPP lesions (g), most pronounced in the KC adjacent to pustules (h). Representative images from 6 GPP, PV and NN donors used (Supplemental Figure 1). DAB with hematoxylin counterstain. Scale bar 100µm.

Article Snippet: Immunohistochemistry was performed on formalin fixed and paraffin embedded human skin biopsies sectioned at 5 μm, and then immunohistochemically stained after antigen retrieval using polyclonal goat anti-IL-36α (1μg/ml, AF1078, R&D Systems), IL-36γ (2.5μg/ml, rat, Y-12, Santa Cruz Biotech), polyclonal goat anti-CXCL8 (5μg/ml, AF2008, R&D Systems), polyclonal rabbit anti-cathepsin G (5μg/ml, NBP233498, Novus Biologicals), neutrophil elastase (1μg/ml, mouse, NP57, Dako), proteinase 3 (1μg/ml, EPR6277, LSBio), and matched isotype control antibodies.

Techniques: Expressing

Journal: The Journal of allergy and clinical immunology

Article Title: IL-1 and IL-36 are dominant cytokines in generalized pustular psoriasis

doi: 10.1016/j.jaci.2016.08.056

Figure Lengend Snippet: Exposure to NETs for 1 hour (a–d) significantly increased the ability of full-length (FL)-IL-36γ to induce CXCL1 and IL8 expression by KC more than 3-fold compared with untreated FL-IL-36γ (a and c), suggesting that enzyme activity within the NETs caused FL-IL-36γ activation. Truncated (T)-IL-36 as positive control for IL-36 activity. Purified neutrophil elastase (NE, e–h) and cathepsin G (CG, i–l) activated FL-IL-36α and FL-IL-36γ respectively which was inhibited by the addition of specific inhibitors of elastase (serpin A1) or cathepsin G (serpin A3). Statistical significance determined with Student’s t-test and indicated *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. non-significant.

Article Snippet: Immunohistochemistry was performed on formalin fixed and paraffin embedded human skin biopsies sectioned at 5 μm, and then immunohistochemically stained after antigen retrieval using polyclonal goat anti-IL-36α (1μg/ml, AF1078, R&D Systems), IL-36γ (2.5μg/ml, rat, Y-12, Santa Cruz Biotech), polyclonal goat anti-CXCL8 (5μg/ml, AF2008, R&D Systems), polyclonal rabbit anti-cathepsin G (5μg/ml, NBP233498, Novus Biologicals), neutrophil elastase (1μg/ml, mouse, NP57, Dako), proteinase 3 (1μg/ml, EPR6277, LSBio), and matched isotype control antibodies.

Techniques: Expressing, Activity Assay, Activation Assay, Positive Control, Purification

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36α was induced in UUO kidney and renal TECs. (A) Real-time PCR showing relative renal levels of IL-36 mRNAs in sham control and UUO mice. (B) IHC staining showing IL-36α protein expression in sham control and UUO mice; the arrows indicate IL-36α–positive renal TECs of atrophic tubules. Original magnification, ×400. (C) Quantitative analysis for the IL-36α–positive tubules. Data are means±SEM of nine mice per group. Western blot analysis for IL-36α protein expression in mouse tubular epithelial cells (mTECs) M-1 under (D) mechanically induced pressure, (E) H2O2, or (F) HMGB-1. (G) Semiquantitative IL-36α levels in mTECs M-1 lysates. Western blot analysis for IL-36α protein expression in house tubular epithelial cells (hTECs) HK-2 under (H) mechanically induced pressure, (I) H2O2, or (J) HMGB-1. (K) Semiquantitative IL-36α levels in hTECs HK-2 lysates. Data are means±SEM of three replicative results obtained from in vitro experiments. *P<0.05; **P<0.01; ***P<0.01; #not detectable.

Article Snippet: For IHC analysis, formalin-fixed and paraffin-embedded renal tissue sections were incubated with primary antibodies against F4/80 (MCA497; Serotec, Kidlington, Oxford, United Kingdom), pNF- κ B p65 (3033; Cell Signaling, Danvers, MA), mouse IL-36 α (AF2297; R&D), collagen 4 (1340–01; Southern Biotech, Birmingham, AL), CD3 (A0452; Dako, Carpinteria, CA), or human IL-36 α (AP5234c; Abgent, San Diego, CA) and then, incubated with HRP-conjugated secondary antibodies (Dako).

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

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling facilitated NLRP3 inflammasome activation in renal tissues of UUO mice and renal TECs. (A) Representative Western blots analysis and semiquantitation for NLRP3, IL-18, and IL-1β in renal tissues. (B) Caspase-1 activity determined by an activity assay in renal tissues. (C) ELISA for urinary protein levels of IL-1β in normal (sham control) or pelvic (UUO) urine samples. Data are means±SEM of nine mice per group. M-1 TECs treated with (D) rIL-36α, LPS (2 μg/ml), or saline for 24 hours (representative Western blots and semiquantitation for NLRP3, pro–IL-18, and pro–IL-1β in lysates) or (E) rIL-36α, LPS, or saline for 24 hours and an additional 1 hour of ATP incubation (representative Western blots for IL-18, IL-1β, and caspase-1 p10 in supernatants). (F) Caspase-1 activity determined by an activity assay in lysate. Data are means±SEM of three replicative results obtained from in vitro experiments. *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: For IHC analysis, formalin-fixed and paraffin-embedded renal tissue sections were incubated with primary antibodies against F4/80 (MCA497; Serotec, Kidlington, Oxford, United Kingdom), pNF- κ B p65 (3033; Cell Signaling, Danvers, MA), mouse IL-36 α (AF2297; R&D), collagen 4 (1340–01; Southern Biotech, Birmingham, AL), CD3 (A0452; Dako, Carpinteria, CA), or human IL-36 α (AP5234c; Abgent, San Diego, CA) and then, incubated with HRP-conjugated secondary antibodies (Dako).

Techniques: Activation Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation, In Vitro

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling facilitated NLRP3 inflammasome activation in cultured macrophages. (A) ELISA analysis of IL-1β protein levels in supernatant of murine J774A.1 macrophages incubated for 30 minutes with or without rIL-36α (150 ng/ml), 5.5 hours with or without 0.5 μg/ml LPS, and 30 minutes with or without 5 mM ATP. (B) Representative Western blots and semiquantitation for NLRP3 and pro–IL-1β in lysates of J774A.1 macrophages incubated with or without rIL-36α (150 ng/ml) for 30 minutes and then treated with or without LPS for 6 hours. (C) Representative Western blots for caspase-1 p10 in supernatants of J774A.1 incubated with or without rIL-36α (150 ng/ml), 5.5 hours with or without 0.5 μg g/ml LPS, and 30 minutes with or without 5 mM ATP. (D) ELISA analysis of IL-1β protein levels in supernatant of peritoneal macrophages from IL-36R KO and WT mice incubated for 5.5 hours with or without 1 μg g/ml LPS and 30 minutes with or without 5 mM ATP. (E) ELISA analysis of IL-1β protein levels in supernatant of BMDMs from WT, TLR4 KO, and MyD88 KO mice treated with similar conditions as J774A.1 above. (F) Representative Western blots and semiquantitation for NLRP3 and pro–IL-1β in lysates of BMDMs from WT, TLR4 KO, and MyD88 KO mice treated with or without rIL-36α (150 ng/ml). Data are means±SEM of three replicative results obtained from the experiments. Mϕ, macrophage *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: For IHC analysis, formalin-fixed and paraffin-embedded renal tissue sections were incubated with primary antibodies against F4/80 (MCA497; Serotec, Kidlington, Oxford, United Kingdom), pNF- κ B p65 (3033; Cell Signaling, Danvers, MA), mouse IL-36 α (AF2297; R&D), collagen 4 (1340–01; Southern Biotech, Birmingham, AL), CD3 (A0452; Dako, Carpinteria, CA), or human IL-36 α (AP5234c; Abgent, San Diego, CA) and then, incubated with HRP-conjugated secondary antibodies (Dako).

Techniques: Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling activated NLRP3 inflammasome activation in BMDCs. (A) ELISA analysis of IL-1β protein levels in supernatant of BMDCs from WT, IL-36R KO, and NLRP3 KO mice incubated with or without rIL-36α (150 ng/ml) for 24 hours. (B) Representative Western blots and (C) semiquantitation for NLRP3, pro–IL-1β, and caspase-1 p10 in lysates of BMDCs from WT, IL-36R KO, and NLRP3 KO mice incubated with or without rIL-36α (150 ng/ml) for 24 hours. Data are means±SEM of three replicative results obtained from the experiments. *P<0.01; ***P<0.01; #not detectable.

Article Snippet: For IHC analysis, formalin-fixed and paraffin-embedded renal tissue sections were incubated with primary antibodies against F4/80 (MCA497; Serotec, Kidlington, Oxford, United Kingdom), pNF- κ B p65 (3033; Cell Signaling, Danvers, MA), mouse IL-36 α (AF2297; R&D), collagen 4 (1340–01; Southern Biotech, Birmingham, AL), CD3 (A0452; Dako, Carpinteria, CA), or human IL-36 α (AP5234c; Abgent, San Diego, CA) and then, incubated with HRP-conjugated secondary antibodies (Dako).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 signaling facilitated DC-induced T cells activation in UUO mice and cell models. (A) Flow cytometric analysis showing the percentages of CD4+/CD69+ and CD8+/CD69+ in renal draining lymphoid cells at day 7 after UUO or sham operation. Data are means±SEM of nine mice per group. (B) BMDCs were incubated with or without rIL-36α (150 ng/ml) for 16 hours, pulsed with OVA257–264 or OVA323–339 peptide, and then, cocultured with OT-I CD8 or OT-II CD4 T cells, respectively. After 3 days, the T cell proliferation was measured by [3H]thymidine incorporation. Data are means±SEM of three replicative results obtained from the experiments. (C) Flow cytometric analysis showing expression levels CD40, CD80, and CD86 (within gated CD11c cells) in BMDCs from WT and IL-36R KO mice, which were incubated with or without rIL-36α (150 ng/ml) for 24 hours. Data are means±SEM of three replicative results obtained from the experiments. (D) Flow cytometric analysis showing the numbers of CD11c+ cells in renal draining lymphoid cells at day 7 after UUO or sham operation; data are means±SEM of five mice per group. (E) Flow cytometric analysis showing the CD86+CD11c+ cells in renal draining lymphoid cells at day 7 after UUO or sham operation. Data are means±SEM of five mice per group. rdLN, renal draining lymph node. *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: For IHC analysis, formalin-fixed and paraffin-embedded renal tissue sections were incubated with primary antibodies against F4/80 (MCA497; Serotec, Kidlington, Oxford, United Kingdom), pNF- κ B p65 (3033; Cell Signaling, Danvers, MA), mouse IL-36 α (AF2297; R&D), collagen 4 (1340–01; Southern Biotech, Birmingham, AL), CD3 (A0452; Dako, Carpinteria, CA), or human IL-36 α (AP5234c; Abgent, San Diego, CA) and then, incubated with HRP-conjugated secondary antibodies (Dako).

Techniques: Activation Assay, Incubation, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: IL-36 facilitated Th17 differentiation in UUO mice and in vitro T cell activation. (A) Real-time PCR showing relative renal mRNA levels of IL-23p19 and IL-17A in sham control and day14 UUO mice. Data are means±SEM of nine mice per group. (B) BMDCs were incubated with or without rIL-36α (150 ng/ml) for 16 hours, pulsed with OVA323–339 peptides, and then, cocultured with OT-II CD4 T cells. After 4 days, supernatants were collected, and the production of IL-17A was detected by ELISA. Data are means±SEM of three replicative results obtained from the experiments. (C) Real-time PCR showing mRNA levels of specific transcription factors for different Th cells in OT-II CD4 cocultured with BMDCs (preincubated with or without rIL-36α for 16 hours and then, pulsed with OVA323–339 peptides) for 4 days. Data are means±SEM of three replicative results obtained from the experiments. *P<0.05; **P<0.01; ***P<0.01.

Article Snippet: For IHC analysis, formalin-fixed and paraffin-embedded renal tissue sections were incubated with primary antibodies against F4/80 (MCA497; Serotec, Kidlington, Oxford, United Kingdom), pNF- κ B p65 (3033; Cell Signaling, Danvers, MA), mouse IL-36 α (AF2297; R&D), collagen 4 (1340–01; Southern Biotech, Birmingham, AL), CD3 (A0452; Dako, Carpinteria, CA), or human IL-36 α (AP5234c; Abgent, San Diego, CA) and then, incubated with HRP-conjugated secondary antibodies (Dako).

Techniques: In Vitro, Activation Assay, Real-time Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: IL-36 Signaling Facilitates Activation of the NLRP3 Inflammasome and IL-23/IL-17 Axis in Renal Inflammation and Fibrosis

doi: 10.1681/ASN.2016080840

Figure Lengend Snippet: Proposed mechanistic pathways underlying the pathogenic role of IL-36 in the development of renal TILs. IL-36 may be contributed to the pathogenesis of renal TILs, involving the activation of NLRP3 inflammasome and IL-23/IL-17 axis.

Article Snippet: For IHC analysis, formalin-fixed and paraffin-embedded renal tissue sections were incubated with primary antibodies against F4/80 (MCA497; Serotec, Kidlington, Oxford, United Kingdom), pNF- κ B p65 (3033; Cell Signaling, Danvers, MA), mouse IL-36 α (AF2297; R&D), collagen 4 (1340–01; Southern Biotech, Birmingham, AL), CD3 (A0452; Dako, Carpinteria, CA), or human IL-36 α (AP5234c; Abgent, San Diego, CA) and then, incubated with HRP-conjugated secondary antibodies (Dako).

Techniques: Activation Assay

Journal: International Journal of Molecular Medicine

Article Title: Regulation of TLR4 expression mediates the attenuating effect of erythropoietin on inflammation and myocardial fibrosis in rat heart

doi: 10.3892/ijmm.2018.3707

Figure Lengend Snippet: Concentrations of TGF-β1, TNF-α, IL-1β, IL-6 and IL-17A in rat serum.

Article Snippet: The antibody targeting β-actin (cat. no. KL002; 1:500) was purchased from Jiancheng Bioengineering Institute of Nanjing (Nanjing, China), and the goat anti-mouse Immunoglobulin G (cat. no. SA00001-1, working dilution: 1:2,000) and Goat anti-rabbit IgG (cat. no. SA00001-2, working dilution: 1:2,000) horseradish peroxidase (HRP)-conjugated secondary antibodies were from ProteinTech Group, Inc. ELISA kits for the detection of TGF-β1 (cat. no. ELR-TGFb1-1), IL-1β (cat. no. ELR-IL1b-1), IL-6 (cat. no. ELR-IL6-1), IL-17A, and TNF-α (cat. no. ELR-TNFa-1) were purchased from RayBiotech, Inc. (Atlanta, GA, USA).

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Regulation of TLR4 expression mediates the attenuating effect of erythropoietin on inflammation and myocardial fibrosis in rat heart

doi: 10.3892/ijmm.2018.3707

Figure Lengend Snippet: Effects of EPO overexpression on the serum levels of inflammatory mediators in rats that underwent AAC. ELISA was employed to determine the levels of inflammatory mediators (A) TGF-β1, (B) TNF-α, (C) IL-1β, (D) IL-6 and (E) IL-17A in the rat serum in each group. The data are expressed as the mean ± standard error of the mean (n=6 per group). * P<0.05 vs. the control group; # P<0.05 vs. the AAC only group; and & P<0.05 vs. the AAC + EPO group. EPO, erythropoietin; AAC, abdominal aortic constriction; TGF, transforming growth factor; TNF, tumor necrosis factor; IL, interkleukin; TLR4, Toll-like receptor 4.

Article Snippet: The antibody targeting β-actin (cat. no. KL002; 1:500) was purchased from Jiancheng Bioengineering Institute of Nanjing (Nanjing, China), and the goat anti-mouse Immunoglobulin G (cat. no. SA00001-1, working dilution: 1:2,000) and Goat anti-rabbit IgG (cat. no. SA00001-2, working dilution: 1:2,000) horseradish peroxidase (HRP)-conjugated secondary antibodies were from ProteinTech Group, Inc. ELISA kits for the detection of TGF-β1 (cat. no. ELR-TGFb1-1), IL-1β (cat. no. ELR-IL1b-1), IL-6 (cat. no. ELR-IL6-1), IL-17A, and TNF-α (cat. no. ELR-TNFa-1) were purchased from RayBiotech, Inc. (Atlanta, GA, USA).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay