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: eLife

Article Title: Cleavage activates Dispatched for Sonic Hedgehog ligand release

doi: 10.7554/eLife.31678

Figure Lengend Snippet: ( A ) NIH3T3 cells expressing DispHA were treated with increasing concentrations of Furin Inhibitor I (10, 25, 50, 75 and 100 μM). The bracket indicates the 250 kDa fraction. Graph shows Disp densitometry analysis normalized to Kinesin for two independent experiments. Normalized signal intensity for each DispHA species in treated conditions is shown relative to vehicle control intensity, which was set to 1. ( B ) CRISPR/Cas9 generated knockout lines for Furin, PACE4, PCSK5 and PCSK7 were transfected with V5DispHA-expression vector, and formation of the ~30 kDa V5 cleavage fragment was monitored by western blot of cell lysates from Clonal line 1. Furin and PCSK7 protein levels were examined by western blot. PACE4 and PCSK5 mutations were confirmed by deep sequencing as in . ( C ) Lysates from cells co-expressing wild type or CS mutant V5DispHA and Furin-Myc proteins were examined for Disp cleavage by western blot for the V5 fragment. Co-expression of Furin-Myc reduced total Disp signal (top panel). Gain equalization of the V5 signal in Furin-Myc expressing cell lysates is shown for comparison. ( D ) Furin-Myc and V5DispHA were co-expressed in HEK293T cells and Furin-Myc was immunoprecipitated from lysates using anti-Myc. Input (left) and immunoprecipitates (right) are shown. ( E ) Wild type and CS mutant V5DispHA proteins were expressed in LoVo (lacking Furin) or HCT-15 (control) colon carcinoma cells and lysates were analyzed by western blot. Re-expression of Furin in LoVo cells rescued cleavage (lane 4 compared to 2). Kinesin and Tubulin are the loading controls for western blots. 10.7554/eLife.31678.008 Figure 3—source data 1. Data for .

Article Snippet: Transfected construct ( H. sapiens ) , pCMV6 - huFurin Myc DDK , This paper , Origene, RC204279 , .

Techniques: Expressing, CRISPR, Generated, Knock-Out, Transfection, Plasmid Preparation, Western Blot, Sequencing, Mutagenesis, Immunoprecipitation

Journal: eLife

Article Title: Cleavage activates Dispatched for Sonic Hedgehog ligand release

doi: 10.7554/eLife.31678

Figure Lengend Snippet: ( A ) Genotype of PC family knockout clones. Red arrows indicate the Cas9 cleavage site. sgRNA sequence (5’ to 3’) is shown for each targeted gene along with corresponding deletions (black dashes) and insertions (red letters) identified by deep sequencing of knockout clonal lines. Insertion/deletion sizes are indicated in parenthesis and resulting premature truncation amino acid is shown in red. ( B ) CRISPR/Cas9 generated knockout lines for Furin, PACE4, PCSK5 and PCSK7 were transfected with V5DispHA-expression vector, and formation of the ~30 kDa V5 cleavage fragment was monitored by western blot of cell lysates from Clonal line 2. Furin and PCSK7 protein levels were examined by western blot. Kinesin is loading control. PACE4 and PCSK5 were assessed by deep sequencing as in A.

Article Snippet: Transfected construct ( H. sapiens ) , pCMV6 - huFurin Myc DDK , This paper , Origene, RC204279 , .

Techniques: Knock-Out, Clone Assay, Sequencing, CRISPR, Generated, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: eLife

Article Title: Cleavage activates Dispatched for Sonic Hedgehog ligand release

doi: 10.7554/eLife.31678

Figure Lengend Snippet: ( A ) Lysates from Drosophila Cl8 cells treated with control or disp dsRNA or transfected with pAc-dispHA were analyzed by western blot using anti-dDisp. Actin is the loading control. ( A’ ) Endogenous dDisp150 and dDisp110 were specifically immunoprecipitated with anti-dDisp from wing imaginal disc lysate. ( B ) V5dDispHA was expressed in Cl8 cells and lysates were analyzed by western blot to confirm generation of the V5 fragment. Kinesin is the loading control. ( C ) Lysates were prepared from Cl8 cells expressing Δ206–238 (ΔCS) dDispHA protein and analyzed by western blot. Lysates were treated with Endo H or PNGase F. Kinesin is the loading control. ( D–F ) Wild type or ΔCS dDispHA proteins were expressed dorsally in wing imaginal discs using apterous-GAL4 . Representative male wings are shown. ( G–I ) WT or ΔCS V5dDispHA proteins (magenta) were expressed with HhGFP (green) in salivary glands using SGS-GAL4 . Maximum intensity projections of basolateral and basal optical sections of salivary glands are shown. Square in the GFP images indicates zoom area.

Article Snippet: Transfected construct ( H. sapiens ) , pCMV6 - huFurin Myc DDK , This paper , Origene, RC204279 , .

Techniques: Transfection, Western Blot, Immunoprecipitation, Expressing

Journal: eLife

Article Title: Cleavage activates Dispatched for Sonic Hedgehog ligand release

doi: 10.7554/eLife.31678

Figure Lengend Snippet:

Article Snippet: Transfected construct ( H. sapiens ) , pCMV6 - huFurin Myc DDK , This paper , Origene, RC204279 , .

Techniques: CRISPR, Transfection, Construct, Clone Assay, Plasmid Preparation, Software, Western Blot, Luciferase, Reporter Assay

Journal: Advanced Science

Article Title: Semaphorin 3E–Plexin D1 Axis Drives Lung Fibrosis through ErbB2‐Mediated Fibroblast Activation

doi: 10.1002/advs.202415007

Figure Lengend Snippet: Furin inhibitor suppresses differentiation, proliferation, and migration of fibroblasts in IPF. A) Western blot analysis of Furin levels in primary human lung fibroblasts (PHLFs) exposed to varying concentrations of TGF‐β1 for 48 h. B) Western blot analysis of P61‐Sema3E, Fibronectin, Col1a1, and α‐SMA expression in PHLFs treated with different concentrations of the Furin inhibitor Hexa‐D‐arginine following TGF‐β1 induction for 48 h. C) Representative images of EdU staining in PHLFs treated with Hexa‐D‐arginine after TGF‐β1 induction for 48 h. D) Representative images from the Transwell assay in PHLFs treated with Hexa‐D‐arginine following TGF‐β1 induction for 48 h. Images were captured at ×200 magnification. E,G) Histological assessment of lung fibrosis severity in mice following BLM induction. (E) Left panel: Representative lung sections stained with H&E, Masson's trichrome, and Sirius Red to evaluate fibrosis severity. (G) Right panel: Bar graph quantifying fibrosis severity scores (Saline: n = 5, BLM: n = 5, BLM + Hexa‐D‐arginine: n = 5). F) Schematic representation of the experimental design. Mice received intratracheal instillation of 2 mg kg −1 BLM and were administered intraperitoneal injections of Hexa‐D‐arginine starting on day 7 until the end of the experiment. H) Quantification of hydroxyproline content in lung tissues. I) Western blot analysis of Fibronectin, Col1a1, and α‐SMA protein expression in lung homogenates from the indicated mouse groups (Saline: n = 5, BLM: n = 5, BLM + Hexa‐D‐arginine: n = 5). Data are presented as the mean ± SEM from three independent experiments. Statistical significance was determined using one‐way ANOVA (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The Furin inhibitor Hexa‐D‐arginine (HY‐P1028, MedChemExpress, China) was obtained from MedChemExpress.

Techniques: Migration, Western Blot, Expressing, Staining, Transwell Assay, Saline