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