resource source identifier antibodies anti il 1b r d (Virogen Inc)

Virogen Inc resource source identifier antibodies anti il 1b r d

Figure 1. C1-27 Limits NLRP3 Inﬂammasome Activation (A) Inhibition of GSTO1-1-catalyzed 4-nitrophenacyl glutathione reduction by C1-27 dose dependently. The structure of C1-27 is displayed (n = 3). (B and C) ELISA of <t>IL-1b</t> (B) and TNF-⍺(C) in supernatants from BMDMs primed with 3 h LPS, incubated with 5 mM C1-27 for 45 min, and stimulated with 5 mM ATP for 45 min (n = 3). (D) Immunoblots corresponding to BMDMs treated as in (B) and (C) (n = 3). (E and F) ELISA of IL-1b (E) and TNF-⍺(F) in supernatants from BMDMs primed with LPS, incubated with 5 mM C1-27, and stimulated with 10 mM nigericin (n = 3). (G) Immunoblot corresponding to BMDMs treated as in (E) and (F) (n = 3). (H) Pyroptosis of BMDMs treated from (E) and (F) measured by secretion of lactate dehydrogenase (LDH) into the supernatant. % Cytotoxicity was calculated relative to total lysis control of unstimulated cells (n = 3). (I) Immunoblot of BMDMs pre-treated with 5 mM C1-27 and stimulated with LPS for indicated times (n = 3).

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Journal: Cell reports

Article Title: Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation.

doi: 10.1016/j.celrep.2019.08.072

Figure Lengend Snippet: Figure 1. C1-27 Limits NLRP3 Inﬂammasome Activation (A) Inhibition of GSTO1-1-catalyzed 4-nitrophenacyl glutathione reduction by C1-27 dose dependently. The structure of C1-27 is displayed (n = 3). (B and C) ELISA of IL-1b (B) and TNF-⍺(C) in supernatants from BMDMs primed with 3 h LPS, incubated with 5 mM C1-27 for 45 min, and stimulated with 5 mM ATP for 45 min (n = 3). (D) Immunoblots corresponding to BMDMs treated as in (B) and (C) (n = 3). (E and F) ELISA of IL-1b (E) and TNF-⍺(F) in supernatants from BMDMs primed with LPS, incubated with 5 mM C1-27, and stimulated with 10 mM nigericin (n = 3). (G) Immunoblot corresponding to BMDMs treated as in (E) and (F) (n = 3). (H) Pyroptosis of BMDMs treated from (E) and (F) measured by secretion of lactate dehydrogenase (LDH) into the supernatant. % Cytotoxicity was calculated relative to total lysis control of unstimulated cells (n = 3). (I) Immunoblot of BMDMs pre-treated with 5 mM C1-27 and stimulated with LPS for indicated times (n = 3).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-IL-1b R&D Cat# AF401-NA Anti-caspase-1 Adipogen Cat# AG-20B-0042-C100 Anti-NLRP3 Cell Signaling Cat# D4D8T Anti-b-actin Sigma Cat# 4267 Anti-ASC Santa Cruz Cat# Sc-22514-r Anti-NEK7 Abcam Cat# Ab133514 Anti-GSTO1-1 Genetex Cat# GTX118439 Anti-GSH Virogen Cat# 101-A-100 Anti-HA Sigma Cat# H3663 Anti-FLAG Sigma Cat# F1804 Anti-phospho-p38 Cell Signaling Cat# 9211 Anti-IkBa Cell Signaling Cat# 44D4 Anti-phospho-p65 Cell Signaling Cat# 93H1 Bacterial and Virus Strains E.coli BL21(DE3) Novagen Cat# 69450 S. typhimurium UK-1 strain Dr. Sinéad Corr, TCD N/A Chemicals, Peptides, and Recombinant Proteins ATP Invivogen Cat# tlrl-atpl Nigericin Invivogen Cat# tlrl-nig Poly (dA:dT) Invivogen Cat# tlrl-patn C1-27 Prof. Nouri Neamati, University of Michigan N/A Gentamicin Sigma Cat# G1397 GSH-EE Santa Cruz Cat# 118421-50-4 ProLong Gold antifade reagent with DAPI Biosciences Cat# P36931 Ultrapure rough LPS from E. coli, serotype EH100 Alexis Cat# ALX-581-010-L001 HA peptide Sigma Cat# 116669750001 FuGENE HD Transfection Reagent Promega Cat# E2311 Strataclean Resin Agilent Technologies Cat# 400714 Dextran sulfate sodium salt (DSS) Colitis Grade MP Biomedicals Cat# 0216011010 S-(4-nitrophenacyl)glutathione Prof. Philip Board, ANU N/A Disuccinimidyl suberate ThermoFisher Cat# 21655 Lipofectamine 2000 ThermoFisher Cat# 11668030 Lipofectamine RNAiMAX ThermoFisher Cat# 13778030 Critical Commercial Assays Mouse IL-1b DuoSet ELISA R&D Systems Cat# DY401 Mouse TNFa DuoSet ELISA R&D Systems Cat# DY410 Human IL-1b DuoSet ELISA R&D Systems Cat# DY201 Mouse IL-1b Quantikine ELISA R&D Systems Cat# MLB00C Mouse IL-1b ELISA kit Abcam Cat# ab216165 Cytotox 96 non-radioactive cytotoxicity assay Promega Cat# G1780 Quikchange II site directed mutagenesis kit Agilent Technologies Cat# 200521 (Continued on next page) e1 Cell Reports 29, 151–161.e1–e5, October 1, 2019

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

Figure 3. C1-27 Is Active In Vivo and Reduces Inﬂammasome Activation Associ- ated with DSS-Induced Colitis and EAE Development Mice were treated with azoxymethane (AOM) 7.5 mg/kg intraperitoneally (i.p.) at day 3. On day 0, DSS 2.5% was included in drinking water. C1-27 was introduced by daily i.p. injection at a dose of 50 mg/kg in 40% cyclodextrin from day 1 until day 7. Mice were sacriﬁced and samples collected on day 8. (A) Serum IL-18 levels (n = 5–6). (B) Colon culture supernatant IL-18 levels (n = 5–6). (C) Colon culture supernatant IL-1b levels (n = 5–6). (D) Liver GSTO1-1 activity with S-(4-nitrophenacyl) glutathione (4NPG; n = 8–12). (E) Clinical score after EAE induction of C57BL/6 mice injected daily with 40% cyclodextrin/PBS vehicle control or 50 mg/kg C1-27 (n = 7). (F) C57BL/6 mice treated in (E) were weighed daily. Percentage weight was calculated from starting weight of mice (n = 7). Data are mean ± SEM; p values calculated using Student’s t test or one-way ANOVA with Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Journal: Cell reports

Article Title: Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation.

doi: 10.1016/j.celrep.2019.08.072

Figure Lengend Snippet: Figure 3. C1-27 Is Active In Vivo and Reduces Inﬂammasome Activation Associ- ated with DSS-Induced Colitis and EAE Development Mice were treated with azoxymethane (AOM) 7.5 mg/kg intraperitoneally (i.p.) at day 3. On day 0, DSS 2.5% was included in drinking water. C1-27 was introduced by daily i.p. injection at a dose of 50 mg/kg in 40% cyclodextrin from day 1 until day 7. Mice were sacriﬁced and samples collected on day 8. (A) Serum IL-18 levels (n = 5–6). (B) Colon culture supernatant IL-18 levels (n = 5–6). (C) Colon culture supernatant IL-1b levels (n = 5–6). (D) Liver GSTO1-1 activity with S-(4-nitrophenacyl) glutathione (4NPG; n = 8–12). (E) Clinical score after EAE induction of C57BL/6 mice injected daily with 40% cyclodextrin/PBS vehicle control or 50 mg/kg C1-27 (n = 7). (F) C57BL/6 mice treated in (E) were weighed daily. Percentage weight was calculated from starting weight of mice (n = 7). Data are mean ± SEM; p values calculated using Student’s t test or one-way ANOVA with Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Techniques: In Vivo, Activation Assay, Injection, Activity Assay, Control

Figure 5. GSTO1-1 Enhances NLRP3 Inﬂammasome Reconstitution and NLRP3 Activation Is Limited by Exogenous GSH (A) ELISA of IL-1b secreted by HEK293T cells transiently transfected with plasmids encoding pro-caspase-1, pro-IL-1b, NLRP3, ASC, NEK7, and GSTO1-1 and stimulated with 10 mM nigericin. DNA concentration was kept constant using the relevant empty vector control (n = 3). (B) Immunoblot of HEK293T cells transfected in (A) (n = 3). (C) ELISA of IL-1b in the supernatants from BMDMs primed with LPS for 3 h, incubated with GSH-EE (1 mM, 5 mM, or 10 mM) for 45 min, and stimulated with 5 mM ATP for 45 min (n = 3). (D) Immunoblot of BMDMs treated in (C) (n = 3). Data are mean ± SEM; p values calculated using one-way ANOVA with Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01).

Journal: Cell reports

Article Title: Glutathione Transferase Omega-1 Regulates NLRP3 Inflammasome Activation through NEK7 Deglutathionylation.

doi: 10.1016/j.celrep.2019.08.072

Figure Lengend Snippet: Figure 5. GSTO1-1 Enhances NLRP3 Inﬂammasome Reconstitution and NLRP3 Activation Is Limited by Exogenous GSH (A) ELISA of IL-1b secreted by HEK293T cells transiently transfected with plasmids encoding pro-caspase-1, pro-IL-1b, NLRP3, ASC, NEK7, and GSTO1-1 and stimulated with 10 mM nigericin. DNA concentration was kept constant using the relevant empty vector control (n = 3). (B) Immunoblot of HEK293T cells transfected in (A) (n = 3). (C) ELISA of IL-1b in the supernatants from BMDMs primed with LPS for 3 h, incubated with GSH-EE (1 mM, 5 mM, or 10 mM) for 45 min, and stimulated with 5 mM ATP for 45 min (n = 3). (D) Immunoblot of BMDMs treated in (C) (n = 3). Data are mean ± SEM; p values calculated using one-way ANOVA with Sidak’s multiple comparisons test (*p < 0.05, **p < 0.01).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Transfection, Concentration Assay, Plasmid Preparation, Control, Western Blot, Incubation

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