goat anti mouse il 1b primary antibody  (R&D Systems)

Journal: Frontiers in immunology

Article Title: Inflammasome modulation with P2X7 inhibitor A438079-loaded dressings for diabetic wound healing.

doi: 10.3389/fimmu.2024.1340405

Figure Lengend Snippet: FIGURE 3 In vitro evaluation of A438079-loaded films. (A) ELISA quantification of supernatant IL-1b released from J774DUAL macrophages left untreated or primed for 4 hours with 1 µg/mL LPS and stimulated for 45 minutes with 3 mM ATP. Cells were treated for the final 30 minutes of priming with conditioned media exposed to A438079-loaded silk films for 4 hours. Results are given as mean ± standard error with statistics calculated by two- way ANOVA and Fisher’s LSD. ****p<0.0001. (B) Quantification of perinuclear ASC specks from LPS-primed J774DUAL macrophages stimulated for 45 minutes with 3 mM ATP with or without 30 minutes pre-treatment with conditioned media exposed to A438079-loaded silk films. Results are given as mean ± standard error with statistics calculated by one-way ANOVA with Fisher’s LSD. ***p<0.001. (C) Representative confocal micrographs of J774DUAL macrophages immunostained for ASC (red) and stained for DNA with DAPI (blue) and F-actin with phalloidin (green). Perinuclear ASC specks are indicated with white arrows and shown with enhanced visibility in zoomed fields. Scale bars represent 50 µm in the first two rows and 25 µm in the zoomed field.

Article Snippet: Sections were blocked with 5% bovine serum albumin (BSA) in TBS/0.1% Tween-20 for 1 hour at room temperature and incubated overnight with goat anti-mouse IL-1b primary antibody (R&D Systems, AF-401-NA, 1:100) in 5% BSA in TBS/0.1% Tween-20 at 4°C overnight.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Staining

Journal: Cell reports

Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting.

doi: 10.1016/j.celrep.2024.113700

Figure Lengend Snippet: Figure 1. Fasting human volunteers have elevated arachidonic acid (AA) levels (A) Peripheral blood mononuclear cells were iso- lated from the volunteers and IL-1b production measured by ELISA before and after refeeding (n = 16). (B) Plasma AA was measured in the same volun- teers before and after fasting (n = 21).

Article Snippet: Gels were loaded with 20 mL of the sample per lane, with a final protein mass of 10 mg. Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1b (goat) (AF-401, R&D Systems) 1 in 1000; b-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Cell reports

Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting.

doi: 10.1016/j.celrep.2024.113700

Figure Lengend Snippet: Figure 3. AA inhibits the NLRP3 inflamma- some (A and B) IL-1b production in LPS-primed BMDMs after 1 h stimulation with 10 mM nigericin with or without 40 mM AA added after priming (A) or during priming (B). (C‒F) Cellular viability (C and E) and IL-1b (D and F) produced by LPS-primed WT BMDMs after stimula- tion with nigericin (10 mM, 1 h) (C and D) or ATP (5 mM, 30 min) (E and F) in presence of 40 mM AA. (G‒I) Cellular viability (G), ASC speck quantification (H), and IL-1b (I) produced by WT BMDMs primed with Pam3CSK4 (200 ng/mL, 4 h) after stimulation with palmitic acid (PA; 1 mM, 16 h) in presence of 40 mM AA. (J‒L) Cellular viability (J), ASC speck quantification (K), and IL-1b (L) produced by THP-1 cells (differen- tiated with PMA, 200 ng/mL for 24 h followed by 24 h washout) primed with Pam3CSK4 (200 ng/mL, 4 h) after stimulation with PA (500 mM, 24 h) in presence of 40 mM AA. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way analysis of variance with Tukey’s multiple comparison test or Student’s unpaired t test). n.s., not significant. Data are from at least three inde- pendent experiments (mean and SEM).

Article Snippet: Gels were loaded with 20 mL of the sample per lane, with a final protein mass of 10 mg. Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1b (goat) (AF-401, R&D Systems) 1 in 1000; b-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems).

Techniques: Produced, Comparison

Journal: Cell reports

Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting.

doi: 10.1016/j.celrep.2024.113700

Figure Lengend Snippet: Figure 4. Individual eicosanoids have little ef- fect on NLRP3 activity (A‒D) Cell death (A and C), and IL-1b (B and D) produced by LPS-primed (200 ng/mL for 3 h) WT and Nlrp3/ BMDMs in response to 1 h stimulation with 1 mM misoprostol, PGD2, PGE2, PGF2a, and PGI2. (E‒H) Cell death (E and G) and IL-1b (F and H) pro- duced by LPS-primed WT BMDMs in response to 1 h stimulation with 10 mM nigericin in presence of increasing concentrations of PGF2a (E and F) and PGE2 (G and H). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 in comparison to untreated controls unless indicated otherwise (one-way analysis of variance with Tu- key’s multiple comparison test). Dashed line repre- sents the assay detection limit. Data are from three (A‒D) or four (E‒H) independent experiments (mean and SEM).

Article Snippet: Gels were loaded with 20 mL of the sample per lane, with a final protein mass of 10 mg. Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1b (goat) (AF-401, R&D Systems) 1 in 1000; b-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems).

Techniques: Activity Assay, Produced, Comparison

Journal: Cell reports

Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting.

doi: 10.1016/j.celrep.2024.113700

Figure Lengend Snippet: Figure 5. Cyclooxygenase (COX) activity regulates NLRP3 activity but not NLRC4 (A‒H) Cellular viability (A and D), IL-1b (B and E), and IL-18 (C) produced by LPS-primed (200 ng/mL for 3 h) WT and Nlrp3/ BMDMs in response to NLRP3 stimulant nigericin (10 mM, 1 h) or ATP (5 mM, 30 min) in presence of COX inhibitors indometacin (100 mM) or celecoxib (10 mM). (F‒I) Cellular viability (F), IL-1b (G), IL-18 (H), and PGE2 (I) produced by unprimed WT and Nlrp3/ BMDMs in response to S. Typhimurium infection (MOI 10, 2 h) in presence of COX inhibitors. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way analysis of variance with Tukey’s multiple comparison test). Data are from three independent experiments (mean and SEM).

Article Snippet: Gels were loaded with 20 mL of the sample per lane, with a final protein mass of 10 mg. Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1b (goat) (AF-401, R&D Systems) 1 in 1000; b-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems).

Techniques: Activity Assay, Produced, Infection, Comparison

Journal: Cell reports

Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting.

doi: 10.1016/j.celrep.2024.113700

Figure Lengend Snippet: Figure 6. PLC is required for canonical NLRP3 activity (A and B) Cell death (A) and IL-1b production (B) in LPS-primed (200 ng/mL for 3 h) WT BMDMs in response to 1 h stimulation with 10 mM nigericin in presence of 1 (+) or 10 mM (++)PLA2 inhibitors ASB1414780 and NAAA or PLC inhibitor U-73122. (C and D) Cell death (C) and IL-1b (D) produced by LPS-primed WT BMDMs stimulated for 1 h with 10 mM nigericin in presence of increasing concen- trations of PLC inhibitor U-73122. (E and F) Cell death (E) and IL-1b (F) produced by LPS-primed WT BMDMs stimulated for 30 min with 5 mM ATP in presence of 1 mM ASB14780 (PLA2 inhibitor) or 1 mM U-73122 (PLC inhibitor). (G) PLC activity of LPS-primed WT BMDMs stimu- lated with 10 mM nigericin for 1 h in presence of U-73122 or 40 mM AA. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (one-way analysis of variance with Tukey’s multiple comparison test or Student’s unpaired t test). Data are from three (E–G) or four (A‒D) independent experiments (mean and SEM).

Article Snippet: Gels were loaded with 20 mL of the sample per lane, with a final protein mass of 10 mg. Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1b (goat) (AF-401, R&D Systems) 1 in 1000; b-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems).

Techniques: Activity Assay, Produced, Comparison

Journal: Cell reports

Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting.

doi: 10.1016/j.celrep.2024.113700

Figure Lengend Snippet: Figure 7. AA inhibits PKC, PKD, and JNK (A and B) Cell death (A) and IL-1b production (B) in THP-1 cells primed with LPS (200 ng/mL, 3 h) fol- lowed by stimulation with nigericin (10 mM, 1 h) in presence of PKC inhibitor (sotrastaurin, 10 mM), PKD inhibitor (CRT006601, 10 mM), JNK inhibitor (SP600125, 10 mM), AA (40 mM), or LCA (30 mM). (C and D) Immunoblots (C) and densitometric quantification (D) of THP-1 cells primed with LPS (200 ng/mL, 3 h) and stimulated with nigericin (10 mM) in presence of AA (40 mM) or LCA (30 mM) for up to 60 min. (E and F) Cell death (E) and IL-1b production (F) in THP-1 cells primed with Pam3CSK4 (200 ng/mL, 4 h) followed by stimulation with PA (500 mM, 16 h) in presence of PKC inhibitor (sotrastaurin, 10 mM), PKD inhibitor (CRT006601, 10 mM), JNK inhibitor (SP600125, 10 mM), AA (40 mM), or LCA (30 mM). (G and H) Quantification of p-JNK (G) and nuclear p- c-Jun (H) in THP-1 cells primed with Pam3CSK4 (200 ng/mL, 4 h) after stimulation with PA (500 mM, 24 h) in presence of 40 mM AA. THP-1 cells were differentiated with PMA (200 ng/ mL) for 24 h followed by 24 h washout prior to the experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 in comparison to nigericin-treated cells (A and B) or PA-treated cells (E and F) unless indicated otherwise (one-way analysis of variance with Tukey’s multiple comparison test). n.s., not significant. Data are from three (C and D), four (A, B, E, and F), or five (G and H) independent experiments (mean and SEM).

Article Snippet: Gels were loaded with 20 mL of the sample per lane, with a final protein mass of 10 mg. Immunoblots were probed using the following primary antibodies: caspase-1 p10 (mouse) (sc-514, Santa Cruz) 1 in 500; IL-1b (goat) (AF-401, R&D Systems) 1 in 1000; b-Actin (mouse) (AB3280, ABCAM) 1 in 2500; NLRP3 (rat) (MAB7578-SP, R&D Systems) 1 in 2000; COX2 (goat) (AF4198, R&D Systems).

Techniques: Western Blot, Comparison