Journal: International Journal of Molecular Sciences

Article Title: Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1β from Human Mast Cells, Augmented by IL-33

doi: 10.3390/ijms24119487

Figure Lengend Snippet: Full-length SARS-CoV-2 spike stimulates IL-1β secretion from human mast cells via TLR4 signaling. LADR mast cells (2.5 × 10 5 cells) were pretreated with anti-TLR2 Ab (2 μg/mL), anti-TLR4 Ab (2 μg/mL), or anti-ACE2 Ab (2 μg/mL) for 1 h and then stimulated with recombinant full-length SARS-CoV-2 S (10 ng/mL) for 1 h and 24 h, respectively. Secretion of chymase ( A ), tryptase ( B ) , and IL-1β ( C ) was determined by specific ELISAs. All conditions were performed in triplicate for each dataset and repeated 3 times ( n = 3). The horizontal brackets indicate the corresponding levels of significance when present ( p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****)).

Article Snippet: LADR Mast Cell Treatments : LADR mast cells were stimulated with recombinant full-length SARS-CoV-2 S (1–10 ng/mL; ab281471, Abcam, Cambridge, UK) or RBD (1–10 ng/mL; ab273065, Abcam, Cambridge, UK), and/or preincubated with the following: (1) anti-TLR2 antibody (2 μg/mL, 1 h; maba2-htlr2, InvivoGen, San Diego, CA, USA), (2) anti-TLR4 antibody (2 μg/mL, 1 h; mabg-htlr4, InvivoGen, San Diego, CA, USA) and (3) anti-ACE2 antibody (2 μg/mL, 1 h; ab108209, InvivoGen, San Diego, CA, USA).

Techniques: Recombinant

Journal: Journal of Biological Methods

Article Title: Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling

doi: 10.14440/jbm.2023.408

Figure Lengend Snippet: AZ617 is a human-specific, TLR4-dependent agonist that activates NFκB. A. Stimulation of human PBMCs, but not mouse splenocytes, with AZ617 for 24 hours effected a concentration-dependent release of NFκB-related cytokines IFNγ, IL-1β, TNFα, and IL-6. Representative graphs generated from 4 independent experiments. B. AZ617 activity was reduced when human PBMCs were pre-treated with an anti-human TLR4 blocking reagent for one hour prior to stimulation. Representative graphs derived from 2 independent experiments. Unpaired t-test **p#x003C;0.01, ***p#x003C;0.001. Error bars represent standard error of the mean (SEM).

Article Snippet: For TLR4-dependency studies, human PBMCs were pre-incubated with 12.5 μg/mL anti-human TLR4 blocking antibody (Invivogen, mabg-htlr4) for 1 hour at 37ºC prior to stimulation with 50 ng/mL AZ617 for an additional 24 hours.

Techniques: Concentration Assay, Generated, Activity Assay, Blocking Assay, Derivative Assay