The inflammasome assay was performed as previously described with some modifications3 (link),4 (link), all experiments were repeated three times. Briefly, plasmids of pCMV-mPro-IL-1β (100 ng, Addgene no. 73953, a gift from C. Stehlik44 ), pCMV-mCaspase1-C-flag (10 ng, Addgene no. 21142, a gift from J. Yuan45 ), pCS2-Myc-NLRC4 (300 ng, a gift from F. Shao), pcDNA3.4-Flag-NAIP5 WT/mutant (300 ng) were cotransfected into HEK293T cells (gifted from X. Xiao) in a 12-well plate. After 24 h of transfection, LFn-FlaA and protective antigen proteins were added to the culture medium to a final concentration of 10 μg ml−1. The same concentrations of LFn-FlaAAA and protective antigen were added to the negative control well. After another 24 h, the transfected cells were gathered in a passive lysis buffer (250 μl per well; Promega no. E194A) and obtained supernatants were subjected to western blot analysis. The detection of cleaved IL-1β (p17) on an immunoblot confirmed the occurrence of active inflammasome complex formation. Antibodies used were: anti-IL-1 (Abcam no. 234437), anti-Flag (Sigma no. F1804), anti-Myc (Cell Signaling no. 2276), anti-Caspase-1 (Cell Signaling no. 24232), antimouse IgG (Cell Signaling no. 7076), antirabbit IgG (Cell Signaling no. 7074S) and anti-β-tubulin (Invitrogen no. MA5–16308-HRP).
Anti il 1
Manufactured by Abcam
Sourced in United States
Anti-IL-1 is a laboratory reagent used for the detection and quantification of interleukin-1 (IL-1) in various biological samples. It is a specialized tool primarily used in research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti caspase 1, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Thermo Fisher Scientific (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anisomycin, by Merck Group (1 mentions)
Anti beclin1, by Abcam (1 mentions)
Triphenyl tetrazolium chloride (ttc), by Merck Group (1 mentions)
Rabbit anti akt, by Abcam (1 mentions)
Horseradish peroxidase linked goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Anti mtor, by Abcam (1 mentions)
Ly294002, by Merck Group (1 mentions)
Anti lc3, by Abcam (1 mentions)
Dab detection kit, by Beyotime (1 mentions)
Anti tlr4, by Abcam (1 mentions)
Anti t akt, by Abcam (1 mentions)
Anti t pi3k, by Abcam (1 mentions)
Anti caspase 3, by Abcam (1 mentions)
Anti p akt, by Abcam (1 mentions)
4 protocols using anti il 1
1
Inflammasome Activation Assay: Unlocking Immune Responses
2
Deltonin Isolation and Characterization
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Deltonin with a purity of over 98% was generated as previously described and its purity determined by high-performance liquid chromatography (>98%) according to Tong et al.1 (link)
Figure 1A shows the chemical structure of deltonin. LY294002, anisomycin and TTC reagents were obtained from Sigma, USA. Assays to detect IL-6, IL-10, TNF-α, and iNOS were provided by Jiancheng Bio. Ins., China. Rabbit anti-Akt, anti-Phospho-Akt (p-Akt), anti-mTOR, anti-Phospho-mTOR (anti-p-mTOR), anti-P38, anti-Phospho-p38 (anti-p-P38), anti-LC3, anti-Beclin-1, anti-microtubule-associated protein (anti-MAP-2), anti-TLR4 and anti-IL-1 were obtained from Abcam, USA). Rabbit anti-GAPDH was purchased from Beijing Zhongshan Jinqiao Bio., China. Horseradish peroxidase linked goat anti-rabbit IgG was provided by Santa Cruz Biotechnology, US.![]()
Deltonin’s chemical structure (
3
Immunohistochemical Staining of IL-1
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Sections were treated with hyaluronidase and goat serum (Beyotime) before immunohistochemical staining. Then the sections were incubated with primary antibody anti‐IL‐1 (1:200; Abcam) at 4°C for 12 h, respectively. After incubation with biotinylated secondary antibodies, sections were detected with a DAB detection kit (Beyotime).
4
Molecular Signaling in Ovarian Tissue
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The protein levels of toll-like receptor 4 (TLR4), phosphatidylinositol 3-kinase (PI3K), AKT serine (AKT), tumor necrosis factor-α (TNF-α), LYN proto-oncogene, Src family tyrosine kinase (LYN), and caspase-3 were determined by western blotting. The total proteins of ovary tissues were extracted with a protein extraction kit (Beijing Baiolaibo Technology Co. LTD, Beijing, China) and a BCA protein assay kit (Boster Biological Technology Co. LTD, Wuhan, China) was used to determine the concentration of total protein. Furthermore, the proteins were separated by 12% SDS-PAGE, transferred onto a PVDF membrane, and blocked with 5% BSA. The membranes were then incubated with anti-TLR4 (1:2000, Abcam), anti-T-PI3K (1:5000, Abcam), anti-p-PI3K (1:5000, Abcam), anti-T-AKT (1:5000, Abcam), anti-p-Akt (1:5000, Abcam), anti-TNF-α (1:2000, Abcam), anti-IL-1 (1:2000, Abcam), anti-IL-6 (1:2000, Abcam), anti-caspase-3 (1:5000, Abcam), anti-cleaved caspase-3 (1:5000, Abcam), anti-LYN (1:5000, Abcam), and anti-GAPDH (1:5000, Abcam) antibodies, respectively, at room temperature overnight. Thereafter, IgG-HRP was added to the membranes and incubated at room temperature for 2 h. After washing five times, the protein bands were visualized with ECL.
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