anti tlr8 ab  (Novus Biologicals)

Journal: Scientific Reports

Article Title: EV-A71 induced IL-1β production in THP-1 macrophages is dependent on NLRP3, RIG-I, and TLR3

doi: 10.1038/s41598-022-25458-x

Figure Lengend Snippet: TLR3 and TLR8 are involved with EV-A71-induced IL-1β production in THP-1 macrophages. ( A ) Specific TLR3 siRNA or scrambled siRNA was transfected into PMA-primed THP-1 cells for 48 h. Cells were then infected with EV-A71 at the M.O.I. of 2 for 12 h. The expression of TLR3, EV-A71 viral protein VP0, and β-actin was analyzed by Western blot. ( B ) The IL-1β levels in the supernatants were determined by ELISA. ( C ) THP-1 cells were transduced with shNC or shTLR8. The transfected cells were selected and maintained in the puromycin-containing medium. The selected cells were infected with EV-A71 at the M.O.I. of 2 for 12 h. The expression levels of TLR8 and EV-A71 VP0 were examined by Western blot analysis. ( D ) The IL-1β levels in supernatants were assessed by ELISA. ( E ) Wildtype (WT) and NLRP3 KD THP-1 macrophages were transfected with siTLR3 or scrambled siRNA. The transfected cells were infected with EV-A71 at the M.O.I. of 2 for 12 h. The expression levels of TLR3, NLRP3, EV-A71 VP0, and β-actin were determined by Western blot analysis. ( F ) The supernatant was collected and the secreted IL-1β was analyzed by ELISA. Data are expressed as mean value \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SD (** p < 0.01, *** p < 0.001, Student’s unpaired T-test).

Article Snippet: After washed by TBST twice, the membrane was then incubated with anti-EV-A71 3D Ab (1:10,000, was gifted by Professor Shih Shin-Ru), anti-EV-A71 VP0 (1:1000, Millipore, MA, USA), anti-IL-1β Ab (1:1000, Cell Signaling Technology, MA, USA), anti-NLRP3 Ab (1:1000, Cell Signaling Technology, MA, USA), anti-ASC Ab (1:2000) (1:1000, Santa Cruz Biotechnology, TX, USA), anti-caspase-1 Ab (1:1000, Santa Cruz Biotechnology, TX, USA), anti-caspase-8 Ab (1:1000, Cell Signaling Technology, MA, USA), Anti-RIG-I Ab (1:1000, Enzo Life Sciences, NY, USA), anti-TLR3 Ab (1:1000, Abcam, CAMB, UK) and anti-TLR8 Ab (1:1000) (Invitrogen, CA, USA)., The unbound antibodies were then removed by washing with TBST three times.

Techniques: Transfection, Infection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Transduction

Journal: Scientific Reports

Article Title: EV-A71 induced IL-1β production in THP-1 macrophages is dependent on NLRP3, RIG-I, and TLR3

doi: 10.1038/s41598-022-25458-x

Figure Lengend Snippet: NLRP3 and RIG-I are implicated in TLR3 and TLR8 induced IL-1β release in THP-1 macrophages. ( A ) THP-1 macrophages were stimulated by poly(A:U) (0.1, 1 µg) for 12 and 24 h. The IL-1β protein levels were detected by ELISA. ( B ) PMA-primed THP-1 were transfected with TLR3 siRNA and scrambled siRNA for 48 h, and then stimulated by 1 µg poly(A:U) for 12 h. The knockdown efficiency was confirmed by Western blot. The expression levels of IL-1β in the supernatant were analyzed by ELISA. ( C ) PMA-primed THP-1 cells were treated with TLR8 agonists motolimod (5, 10 µM) for 12 and 24 h. The supernatant was collected and the expression of IL-1β was examined by ELISA. ( D ) The shTLR8 transduced THP-1 macrophages were treated with motolimod for 12 h. TLR8 and β-actin in the cell lysates were detected by Immunoblot. IL-1β levels were quantified by ELISA. ( E ) The NLRP3 KD THP-1 macrophage was transfected with poly(A:U), or treated with motolimod for 12 h. The IL-1β levels in the supernatants were examined by ELISA. ( F ) PMA-primed THP-1 was transfected with siRIG-I and scrambled siRNA for 48 h. Then the transfected cells were stimulated by poly(A:U) or motolimod for 12 h. The expression of RIG-I and β-actin was determined by Western blot. The secretion of IL-1β was measured by ELISA. Data are expressed as mean value \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SD (* p < 0.05, *** p < 0.001, Student’s unpaired T-test).

Article Snippet: After washed by TBST twice, the membrane was then incubated with anti-EV-A71 3D Ab (1:10,000, was gifted by Professor Shih Shin-Ru), anti-EV-A71 VP0 (1:1000, Millipore, MA, USA), anti-IL-1β Ab (1:1000, Cell Signaling Technology, MA, USA), anti-NLRP3 Ab (1:1000, Cell Signaling Technology, MA, USA), anti-ASC Ab (1:2000) (1:1000, Santa Cruz Biotechnology, TX, USA), anti-caspase-1 Ab (1:1000, Santa Cruz Biotechnology, TX, USA), anti-caspase-8 Ab (1:1000, Cell Signaling Technology, MA, USA), Anti-RIG-I Ab (1:1000, Enzo Life Sciences, NY, USA), anti-TLR3 Ab (1:1000, Abcam, CAMB, UK) and anti-TLR8 Ab (1:1000) (Invitrogen, CA, USA)., The unbound antibodies were then removed by washing with TBST three times.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Functional Metagenomics: A High Throughput Screening Method to Decipher Microbiota-Driven NF-κB Modulation in the Human Gut

doi: 10.1371/journal.pone.0013092

Figure Lengend Snippet: Cell surface (a.) and intracellular (b.) expression of different TLRs as assessed by flow cytometry using antibodies to TLR2 to TLR9 (from BD Biosciences) in HT-29/kb-seap-25 cells using a FACScalibur (BD Biosciences). Controls are represented in red. c. Effect of various TLRs ligands on reporter gene activity quantified as OD measured at 655 nm by HT-29/kb-seap-25 cells assessed after 24 hours stimulation. Ligands are for TLR1/2: Pam3CSK4 (1 µg/mL); TLR2: Heat Kiled Listeria monocytogenes (10 8 cells /mL); TLR3: Poly(I:C) (10 µg/mL); TLR4: E. coli K12 LPS (10 µg/mL); TLR5: Salmonella typhimurium Flagellin (10 µg/mL); TLR6/2: FSL1 (1 µg/mL); TLR7: Imiquimod (1 µg/mL); TLR8: ssRNA40 (1 µg/mL); TLR9: ODN2006 (5 µM). All ligands are from Invivogen. * = p<0.05.

Article Snippet: TLR5, TLR6 and TLR7 Ab were from Imgenex and TLR8 antibody from Axxora.

Techniques: Expressing, Flow Cytometry, Activity Assay