Journal: Molecules

Article Title: The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense

doi: 10.3390/molecules29184449

Figure Lengend Snippet: Summary of the proteolytic cleavage sites of proteases at the S2′ site. ( A ) Amino acid alignment of the S2′ region of the SARS-CoV-2 S protein. ( B ) S2′-peptides were incubated with TMPRSS2, furin, NE, CatG, and PR3 for 2 h at 37 °C. The hydrolysis of the peptide bonds is summarized in a digestion map (blue bars denote the fragments, and red arrows indicate the cleavage sites). Three independent experiments, n = 3. ( C ) Peptides were incubated with furin in the presence or absence of additional Ca 2+ ions, with a CaCl 2 final concentration of 1.2 mM (left panel). Quantification (right panel). n = 3.

Article Snippet: Human NE (4 μg/mL, neutrophil-derived human NE, PN: 16-14-051200, Lot No. EH 2020-03, Athens Research and Technology, Athens, GA, USA), human CatG (4 μg/mL, neutrophil-derived CatG, PN: 16-14-030107, Lot No. CG 2017-01, Athens Research and Technology, Athens, GA, USA), 4 μg/mL recombinant human furin (4 μL furin, containing 5 mM CaCl 2 based on the company’s production, was added to 94 μL PBS pH 7.4, with a final concentration of 0.2 mM; furin Cat. No. 450-47, Lot No. 1011516, Peprotech, Cranbury, NJ, USA) with or without the addition of CaCl 2 to a final concentration of 1.2 mM, or recombinant human TMPRSS2 (4 μg/mL, Cat. No. CSB-YP023924HU, Batch No. DA0471a3g001, BIOZOL, CUSABIO Technology, LLC, TX, USA) was incubated with 200 μg/mL peptide in PBS pH 7.4 (or in the case of TMPRSS2, Tris pH 7.8, 7.7 mM Tris/HCl and 150 mM NaCl) for 2 h at 37 °C.

Techniques: Incubation, Concentration Assay