Detection of IL-1 beta/IL-1F2 by Western Blot
GlcN inhibits caspase-1 activation and the release of IL-1 beta, IL-18 and ASC. (A–D) J774A.1 macrophages were incubated for 4 h with LPS (1 µg/ml) followed by incubation for 2 h with GlcN. Cells were then incubated with ATP (5 mM, 0.5 h) or infected with E. coli (30 MOI, 1 h). The expression levels of IL-1 beta and IL-18 (A), caspase-1 (C), ASC (D) in the supernatants and IL-1 beta in the cell lysates (B) were analysed by Western blotting. (E) J774A.1 macrophages were incubated for 4 h with LPS (1 µg/ml) followed by incubation for 2 h with GlcN, D-(+)-glucose (Glu), D-glucosamine 3-sulphate (GlcN-3S), D-(+)-galactosamine hydrochloride (GalN) and N-acetyl-D-glucosamine (GlcNAc) for 2 h, followed by incubation with ATP (5 mM) for 0.5 h. The IL-1 beta expression levels in the supernatants were measured by ELISA. (F) J774A.1 macrophages were incubated for 4 h with LPS (1 µg/ml) or PamsCSK4 (1 µg/ml; for non-canonical inflammasome) followed by incubation for 2 h with GlcN, followed by transfection with poly(dA/dT) (2 µg/ml) or LPS (2 µg/ml) for 6 h or by Salmonella infection (30 MOI) for 2 h. The IL-1 beta expression levels in the supernatants were measured by ELISA. The ELISA data are expressed as the mean ± SD of separate experiments as indicated. The Western blotting results are representative of three different experiments and the histogram shows the quantification expressed as the mean ± SD for these three experiments. *, *** and **** indicate a significant difference at the level of p < 0.05, p < 0.001 and p < 0.0001, respectively, compared to NLRP3 activator-treated cells. (One-way ANOVA with Dunnett’s multiple comparisons test). The blots in (A–D) were cropped from different gels; full-length blots are included in the “Supplementary Information”. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30944389), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
CVL reduced NLRP3 inflammasome activation. (A) J774A.1 macrophages were incubated for 5 h with LPS (1 μg/ml) (LPS priming) followed by incubation for 0.5 h with CVL. Cells were then incubated with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), ATP (5 mM, 0.5 h), nigericin (10 μM, 0.5 h), and nano-SiO2 (100 μg/ml, 24 h). (B) LPS-primed J774A.1 macrophages were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml, 24 h), MSU (100 μg/ml, 24 h), and ATP (5 mM, 0.5 h). (C) LPS-primed BMDM were incubated for 0.5 h with CVL followed by incubation with CC (100 μg/ml) for an additional 24 h. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) cells were incubated for 0.5 h with CVL followed by transfection with poly(dA/dT) (2 μg/ml, 6 h), FLA-ST (1 μg/ml, 6 h), MDP (10 μg/ml, 6 h), or LPS (2 μg/ml, 6 h). The levels of IL-1 beta, IL-18, NLRP3, ASC, and caspase-1 in the culture medium were measured by Western blot. The IL-1 beta levels in the supernatants were measured by ELISA. The Western blot results are representative of three different experiments. The ELISA data are expressed as the mean ± SD of three separate experiments. *, **, and *** indicate a significant difference at the level of p < 0.05, p < 0.01, and p < 0.001, respectively, compared to activator-treated cells. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30186288), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
F240B inhibits the NLRP3 inflammasome through autophagy induction. (A, B) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with F240B for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. The levels of IL-1 beta and caspase-1 (A) or NLRP3 and ASC (B) in the supernatants were measured by Western blotting. (C) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B in the presence or absence of 5 mM 3-MA for 3 h. Cells then were incubated with 5 mM ATP or 10 μM nigericin for 0.5 h. The levels of IL-1 beta in the supernatants were measured by ELISA. (D) Wild-type and LC3-knockout J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B for 3 h. Cells then incubated with 5 mM ATP or 10 μM nigericin for 0.5 h. The levels of IL-1 beta in the supernatants were measured by ELISA. The data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p < 0.05 and p < 0.001, respectively. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/33424855), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
ALKBH5 regulates the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) ALKBH5 knockdown enhanced the expression levels of the IL-1 beta, CSF3, TGM2, and SRC transcripts during P. aeruginosa, C. diphtheriae, HSV-1, or HSV-1 ICP34.5 mutant infection. (B to D) ALKBH5 knockdown enhanced the protein level of IL-1 beta during infection by C. diphtheriae (B), P. aeruginosa (C), or the HSV-1 ICP34.5 mutant (D). (E) ALKBH5 overexpression inhibited the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during P. aeruginosa infection as measured by RT-qPCR. (F) ALKBH5 overexpression inhibited the protein level of IL-1 beta during P. aeruginosa infection as measured by Western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
DUSP1 regulates p38 and JNK phosphorylation and the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) DUSP1 knockdown enhanced p38 and JNK phosphorylation during infection by P. aeruginosa, C. diphtheriae, HSV-1, or the HSV-1 ICP34.5 mutant. (B) DUSP1 knockdown enhanced the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during infection by P. aeruginosa, C. diphtheriae, HSV-1, or the HSV-1 ICP34.5 mutant. (C) DUSP1 knockdown enhanced the protein level of IL-1 beta during infection by P. aeruginosa, C. diphtheriae, or the HSV-1 ICP34.5 mutant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
ALKBH5 regulates the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) ALKBH5 knockdown enhanced the expression levels of the IL-1 beta, CSF3, TGM2, and SRC transcripts during P. aeruginosa, C. diphtheriae, HSV-1, or HSV-1 ICP34.5 mutant infection. (B to D) ALKBH5 knockdown enhanced the protein level of IL-1 beta during infection by C. diphtheriae (B), P. aeruginosa (C), or the HSV-1 ICP34.5 mutant (D). (E) ALKBH5 overexpression inhibited the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during P. aeruginosa infection as measured by RT-qPCR. (F) ALKBH5 overexpression inhibited the protein level of IL-1 beta during P. aeruginosa infection as measured by Western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
ALKBH5 regulates the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) ALKBH5 knockdown enhanced the expression levels of the IL-1 beta, CSF3, TGM2, and SRC transcripts during P. aeruginosa, C. diphtheriae, HSV-1, or HSV-1 ICP34.5 mutant infection. (B to D) ALKBH5 knockdown enhanced the protein level of IL-1 beta during infection by C. diphtheriae (B), P. aeruginosa (C), or the HSV-1 ICP34.5 mutant (D). (E) ALKBH5 overexpression inhibited the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during P. aeruginosa infection as measured by RT-qPCR. (F) ALKBH5 overexpression inhibited the protein level of IL-1 beta during P. aeruginosa infection as measured by Western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
DUSP1 regulates p38 and JNK phosphorylation and the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) DUSP1 knockdown enhanced p38 and JNK phosphorylation during infection by P. aeruginosa, C. diphtheriae, HSV-1, or the HSV-1 ICP34.5 mutant. (B) DUSP1 knockdown enhanced the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during infection by P. aeruginosa, C. diphtheriae, HSV-1, or the HSV-1 ICP34.5 mutant. (C) DUSP1 knockdown enhanced the protein level of IL-1 beta during infection by P. aeruginosa, C. diphtheriae, or the HSV-1 ICP34.5 mutant. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
CS inhibits NLRP3 inflammasome activation. (A–D) J774A.1 macrophages were primed with LPS for 5 h and incubated with CS for 0.5 h Cells were stimulated with 10 μM nigericin for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA (A) and Western blot (B). The levels of IL-18 (C) and caspase-1 (D) in the supernatants were measured by Western blot. (E, F) Human THP-1 macrophages or PBMC were primed with LPS for 5 h and incubated with CS for 0.5 h Cells were stimulated with 10 μM nigericin for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA. (G, H) J774A.1 macrophages were primed with LPS for 5 h and incubated with CS for 0.5 h Cells were stimulated with 5 mM ATP for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA (G) and the levels of caspase-1 in the supernatants were measured by Western blot (H). (I) J774A.1 macrophages were primed with LPS or Pam3CSK4 (for non-canonical inflammasome) for 5 h and incubated with CS for 0.5 h Cells were transfected with poly(dA/dT), LPS, MDP or FLA-ST for 6 h or infected with Salmonella for 2 h The levels of IL-1 beta in the supernatants were measured by ELISA. (J) J774A.1 macrophages were primed with LPS for 5 h and incubated with Irbesartan (IS) for 0.5 h Cells were stimulated with 10 μM nigericin for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA. The ELISA data are expressed as the means ± SD of the three separate experiments. The Western blot images are representative results, and the histogram shows the band intensity. *, ** and *** indicate a significant difference at the level of p<0.05, p<0.01 and p<0.001, respectively, compared to nigericin- or ATP-activated cells or as indicated. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35669789), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
ALKBH5 regulates the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) ALKBH5 knockdown enhanced the expression levels of the IL-1 beta, CSF3, TGM2, and SRC transcripts during P. aeruginosa, C. diphtheriae, HSV-1, or HSV-1 ICP34.5 mutant infection. (B to D) ALKBH5 knockdown enhanced the protein level of IL-1 beta during infection by C. diphtheriae (B), P. aeruginosa (C), or the HSV-1 ICP34.5 mutant (D). (E) ALKBH5 overexpression inhibited the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during P. aeruginosa infection as measured by RT-qPCR. (F) ALKBH5 overexpression inhibited the protein level of IL-1 beta during P. aeruginosa infection as measured by Western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
ALKBH5 regulates the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) ALKBH5 knockdown enhanced the expression levels of the IL-1 beta, CSF3, TGM2, and SRC transcripts during P. aeruginosa, C. diphtheriae, HSV-1, or HSV-1 ICP34.5 mutant infection. (B to D) ALKBH5 knockdown enhanced the protein level of IL-1 beta during infection by C. diphtheriae (B), P. aeruginosa (C), or the HSV-1 ICP34.5 mutant (D). (E) ALKBH5 overexpression inhibited the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during P. aeruginosa infection as measured by RT-qPCR. (F) ALKBH5 overexpression inhibited the protein level of IL-1 beta during P. aeruginosa infection as measured by Western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
CS inhibits NLRP3 inflammasome activation. (A–D) J774A.1 macrophages were primed with LPS for 5 h and incubated with CS for 0.5 h Cells were stimulated with 10 μM nigericin for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA (A) and Western blot (B). The levels of IL-18 (C) and caspase-1 (D) in the supernatants were measured by Western blot. (E, F) Human THP-1 macrophages or PBMC were primed with LPS for 5 h and incubated with CS for 0.5 h Cells were stimulated with 10 μM nigericin for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA. (G, H) J774A.1 macrophages were primed with LPS for 5 h and incubated with CS for 0.5 h Cells were stimulated with 5 mM ATP for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA (G) and the levels of caspase-1 in the supernatants were measured by Western blot (H). (I) J774A.1 macrophages were primed with LPS or Pam3CSK4 (for non-canonical inflammasome) for 5 h and incubated with CS for 0.5 h Cells were transfected with poly(dA/dT), LPS, MDP or FLA-ST for 6 h or infected with Salmonella for 2 h The levels of IL-1 beta in the supernatants were measured by ELISA. (J) J774A.1 macrophages were primed with LPS for 5 h and incubated with Irbesartan (IS) for 0.5 h Cells were stimulated with 10 μM nigericin for 0.5 h The levels of IL-1 beta in the supernatants were measured by ELISA. The ELISA data are expressed as the means ± SD of the three separate experiments. The Western blot images are representative results, and the histogram shows the band intensity. *, ** and *** indicate a significant difference at the level of p<0.05, p<0.01 and p<0.001, respectively, compared to nigericin- or ATP-activated cells or as indicated. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35669789), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
ALKBH5 regulates the expression of innate immune response genes during bacterial and viral infections in RAW264.7 cells. (A) ALKBH5 knockdown enhanced the expression levels of the IL-1 beta, CSF3, TGM2, and SRC transcripts during P. aeruginosa, C. diphtheriae, HSV-1, or HSV-1 ICP34.5 mutant infection. (B to D) ALKBH5 knockdown enhanced the protein level of IL-1 beta during infection by C. diphtheriae (B), P. aeruginosa (C), or the HSV-1 ICP34.5 mutant (D). (E) ALKBH5 overexpression inhibited the expression of the IL-1 beta, CSF3, TGM2, and SRC genes during P. aeruginosa infection as measured by RT-qPCR. (F) ALKBH5 overexpression inhibited the protein level of IL-1 beta during P. aeruginosa infection as measured by Western blotting. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36625590), licensed under a CC-BY license. Not internally tested by R&D Systems.
