Journal: International Journal of Molecular Sciences

Article Title: Thymic Stromal Lymphopoietin (TSLP) Is Cleaved by Human Mast Cell Tryptase and Chymase

doi: 10.3390/ijms25074049

Figure Lengend Snippet: Cleavage analysis of TSLP by tryptase and PCSK3. Recombinant human non-glycosylated TSLP (2 μg) was incubated with tryptase (0.2 μg at 37 °C) for 1 h or with PCSK3 (0.88 μg at 37 °C) for 24 h at 37 °C. Aliquots were inactivated by heating for 10 min at 99 °C to stop the cleavage reaction and separated on 16.5% Tris-Tricine gel. The gel was stained with a colloidal Coomassie Brilliant Blue solution.

Article Snippet: Recombinant human furin (PCSK3) (1503-SE, R&D System, Minneapolis, MN, USA), bovine serum albumin, L-glutamine, antibiotic–antimycotic solution (10,000 IU/mL penicillin, 10 mg/mL streptomycin, and 25 μg/mL amphotericin B), RPMI 1640, fetal calf serum (FCS) (endotoxin level < 0.1 EU/mL), 1,4-Piperazinediethanesulfonic acid (PIPES), PBS (14200067, Gibco TM , ThermoFisher Scientific, Waltham, MA, USA), Percoll ® and Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), detoxified lipopolysaccharide (LPS) (from E. coli serotype 0111:B4), IL-4 (Miltenyi Biotec, Bologna, Italy), heparin (PharmaTex Italia, Milan, Italy), and rabbit polyclonal antibody anti-human TSLP (ab109229, Abcam, Milan, Italy) were also obtained.

Techniques: Recombinant, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Thymic Stromal Lymphopoietin (TSLP) Is Cleaved by Human Mast Cell Tryptase and Chymase

doi: 10.3390/ijms25074049

Figure Lengend Snippet: Effects of TSLP cleavage products generated by PCSK3 and tryptase on the release of VEGF-A from human lung macrophages (HLMs). Recombinant human non-glycosylated TSLP (2 μg) was incubated with tryptase (0.2 μg at 37 °C) for 1 h or with PCSK3 (0.88 μg at 37 °C) for 24 h at 37 °C. At the end of the incubation, aliquots of untreated TSLP, tryptase-treated TSLP, and PCSK3-treated TSLP were incubated (18 h, 37 °C) with HLMs in triplicate. At the end of the incubation, the supernatants were collected and VEGF-A concentrations were evaluated by ELISA. The results show the mean ± SD of a typical experiment out of three. ** p < 0.01.

Article Snippet: Recombinant human furin (PCSK3) (1503-SE, R&D System, Minneapolis, MN, USA), bovine serum albumin, L-glutamine, antibiotic–antimycotic solution (10,000 IU/mL penicillin, 10 mg/mL streptomycin, and 25 μg/mL amphotericin B), RPMI 1640, fetal calf serum (FCS) (endotoxin level < 0.1 EU/mL), 1,4-Piperazinediethanesulfonic acid (PIPES), PBS (14200067, Gibco TM , ThermoFisher Scientific, Waltham, MA, USA), Percoll ® and Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA), detoxified lipopolysaccharide (LPS) (from E. coli serotype 0111:B4), IL-4 (Miltenyi Biotec, Bologna, Italy), heparin (PharmaTex Italia, Milan, Italy), and rabbit polyclonal antibody anti-human TSLP (ab109229, Abcam, Milan, Italy) were also obtained.

Techniques: Generated, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay

Journal: npj Viruses

Article Title: K5 polysaccharides inhibit SARS-CoV-2 infection by preventing spike-proteolytic priming

doi: 10.1038/s44298-025-00163-4

Figure Lengend Snippet: a Predominant composition of the disaccharide unit present in sulfated K5 derivatives is shown (see Table – for more details). Disaccharides are represented in their ionic form (sodium salt is the common counterion). b Fraction of spike bound to GAG at increasing concentrations of the indicated GAGs as measured by MST. c Inhibition of the interaction of immobilized heparin with spike by increasing concentrations of the indicated GAGs as measured by SPR. d Inhibition of the binding of spike RBD to immobilizedACE2 at increasing concentrations of the indicated GAGs as measured by SPR. For panel C and D, the responses are plotted as a percentage of the binding of spike in the absence of GAG. e Inhibition of cleavage of a peptide fragment containing the S /S 2 basic domain of spike by increasing concentrations of the indicated GAGs. The peptide was left untreated (- furin) or exposed to furin (25 ng/well) ( + furin) (red points and lanes) and after incubation in the absence and the presence of the GAG, spike cleavage was evaluated by optical density (O.D.) measurements as described in Experimental Procedures. –furin vs + furin: P value < 0.00001. Each point is the mean ± standard deviation (sd) of three to ten separate determinations (see Table for more details).

Article Snippet: Reagents and materials were used as received, unless otherwise mentioned, and were purchased from the following: Human recombinant SARS-CoV-2 Wuhan-Hu-1 spike His-Tag protein and RBD from Sino Biological (#40592-V08B); ACE2 from Acrobiosystem (#AC2-H52H8); Bovine Serum Albumin (BSA) from Merck (#810037); Human recombinant furin from OriGene Technologies Inc. (#TP304279M); Conventional heparin (13.6 kDa - purity ≥95%) from a commercial batch of unfractionated sodium heparin from Laboratori Derivati Organici S.p.A. (#9041-08-1).

Techniques: Inhibition, Binding Assay, Incubation, Standard Deviation