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: PLoS ONE

Article Title: The Proprotein Convertase Furin Contributes to Rhabdomyosarcoma Malignancy by Promoting Vascularization, Migration and Invasion

doi: 10.1371/journal.pone.0161396

Figure Lengend Snippet: A) mRNA levels of all nine proprotein convertases (PCs) were determined by qRT-PCR in 5 Ewing sarcoma, 8 osteosarcoma and 20 rhabdomyosarcoma (RMS) cell lines. Shown are levels relative to GAPDH expression. B) Protein levels of proform and mature furin were assessed by immunoblotting in 20 different RMS cell lines. C) Endogenous furin activity of selected RMS cell lines: Rh36, RD (eRMS), Rh3, Rh4, Rh30, Rh41 and RMS13 (aRMS). Furin activity deficient cells LoVo cells serve as negative control. Furin was captured from cell lysates on anti-furin antibody coated plates and furin activity was measured by addition of the fluorogenic substrate Boc-RVRR-AMC after 6h. Displayed are values normalized by background subtraction.

Article Snippet: Black FluoroNunc 96 well plates (MaxiSorp surface, Nunc, Thermo Scientific) were coated with goat anti human furin antibody (AF1503, R&D Systems) at 10 μg/ml in 50 mM Na 2 CO 3 , pH 9.6 (50 μl/ well) for 8h at RT, protected from light.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Activity Assay, Negative Control

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: Enzymes involved in the processing of proBDNF are altered after pilocarpine-induced SE. Representative Western blots of whole hippocampal protein homogenates from WT mice killed 3 h (left panels) and 24 h (right panels) after the induction of SE or time-matched saline controls. Densitometry analysis of abundance of different cleavage proteins normalized to actin and expressed as the percentage change relative to mean values of the control group (±SEM). A – H , Anti-furin (1:1000; A , B ); anti-plasminogen (1:3000; C , D ); anti-MMP9 (1:2000; E , F ); anti-tPA (1:1000; G , H ). The sample size for 3 h is N = 5 in each group and for 24 h is N = 4 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001; t test.

Article Snippet: The following antibodies and concentrations were used: mouse monoclonal HA.11 clone 16B12 antibody (1:3000; MMS-101P, Covance), mouse monoclonal proBDNF antibody (1:1000; H10001G-MA, GeneCopoeia), rabbit polyclonal to α-2 antiplasmin (1:2000; ab62771, Abcam), rabbit polyclonal furin antibody (1:1000; sc-20801, Santa Cruz Biotechnology), rabbit polyclonal MMP-9 antibody (1:2000; AB13458, Millipore), sheep polyclonal neuroserpin antibody (1:2000; SASMNSP-GF-HT, Molecular Innovations), rabbit polyclonal PAI-1 antibody (1:1000; ASMPAI-GF-HT, Molecular Innovations), rabbit polyclonal plasminogen antibody (1:3000; ASMPLG-GF-HT, Molecular Innovations), sheep polyclonal tPA antibody (1:500; SASTPA-GF-HT, Molecular Innovations), and rabbit polyclonal TIMP-1 antibody (1:1000; AB770, Millipore).

Techniques: Western Blot, Saline, Control

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: Schematic representation of different proteins involved in the cleavage of BDNF through extracellular (left panels) and intracellular (right panels) mechanisms. ProBDNF can be cleaved intracellularly within the endoplasmic reticulum by furin and in regulated secretory vesicles by proconvertase enzymes (PC1/3). ProBDNF can also be cleaved extracellularly by MMPs (−3/−7/−9) or by components of the tPA/plasmin proteolytic cascade. The activity of these proteases is tightly regulated by a number of inhibitors, including PAI-1, which inhibits both extracellular and intracellular cleavage; TIMPs, which inhibit MMPs; and neuroserpin and A2AP, which inhibit the tPA/plasmin proteolytic cascade. Red bars indicate inhibition, and green bars indicate activation.

Article Snippet: The following antibodies and concentrations were used: mouse monoclonal HA.11 clone 16B12 antibody (1:3000; MMS-101P, Covance), mouse monoclonal proBDNF antibody (1:1000; H10001G-MA, GeneCopoeia), rabbit polyclonal to α-2 antiplasmin (1:2000; ab62771, Abcam), rabbit polyclonal furin antibody (1:1000; sc-20801, Santa Cruz Biotechnology), rabbit polyclonal MMP-9 antibody (1:2000; AB13458, Millipore), sheep polyclonal neuroserpin antibody (1:2000; SASMNSP-GF-HT, Molecular Innovations), rabbit polyclonal PAI-1 antibody (1:1000; ASMPAI-GF-HT, Molecular Innovations), rabbit polyclonal plasminogen antibody (1:3000; ASMPLG-GF-HT, Molecular Innovations), sheep polyclonal tPA antibody (1:500; SASTPA-GF-HT, Molecular Innovations), and rabbit polyclonal TIMP-1 antibody (1:1000; AB770, Millipore).

Techniques: Activity Assay, Inhibition, Activation Assay

Journal: Emerging microbes & infections

Article Title: Furin cleavage is required for swine acute diarrhea syndrome coronavirus spike protein-mediated cell - cell fusion.

doi: 10.1080/22221751.2022.2114850

Figure Lengend Snippet: Figure 2. Furin activates SADS-CoV S-mediated cell – cell fusion and S cleavage. (A, B) HEK293T cells were transfected with SADS- CoV S and GFP. After 6 h, transfected cells were treated with the indicated protease inhibitors. Cell – cell fusion was observed by fluorescence microscopy (A). Scale bars, 100 µm. S expression was examined by western blotting (B). (C) Cells were transfected with SADS-CoV S and GFP in the presence or absence of human furin. After 48 h, cell nuclei were stained with DAPI and cell – cell fusion was assessed by fluorescence microscopy. Scale bars, 100 µm. (D) Nuclei in the fuzed cells were counted. Statistical signifi- cance was assessed by Student’s t test. ***, P < 0.001.

Article Snippet: A plasmid encoding human furin containing a C-terminal mycDDK tag was purchased from OriGene (Rockville, MD, USA).

Techniques: Transfection, Microscopy, Expressing, Western Blot, Staining

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: HEK293 FRT furin knockout. HEK293 FRT cells were transfected with plasmids containing genes coding for Cas9 and three furin sgRNAs. Surviving cells were clonally selected by serial dilution in a 96-well plate. Lysates from HEK293 FRT (WT) and four mutant clones were evaluated for furin expression by western blot. Clones 1, 2, and 4 showed no discernable furin expression, and clone 1 (boxed) was selected for further study as ΔFur293.

Article Snippet: Human furin cDNA (product #RC204279) was obtained from OriGene (Rockville, MD, USA) and inserted into the pcDNA5/FRT plasmid for stable transfection into HEK293 FRT cells.

Techniques: Knock-Out, Transfection, Serial Dilution, Mutagenesis, Clone Assay, Expressing, Western Blot

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Cytotoxicity assays. HEK293 FRT and ΔFur293 cells were treated with the anti-transferrin receptor/PE24 RIT HB21-LR in the presence and absence of 1 μM PPCI furin inhibitor. EC 50 (pM) values from four separate paired assays for each condition are plotted. Dotted lines denote the average value for each condition and error bars indicate the standard deviation. Significant P -values ( P <0.05) are reported from a one-way ANOVA performed as described. A representative cytotoxicity assay is shown in Fig. S3 .

Article Snippet: Human furin cDNA (product #RC204279) was obtained from OriGene (Rockville, MD, USA) and inserted into the pcDNA5/FRT plasmid for stable transfection into HEK293 FRT cells.

Techniques: Standard Deviation, Cytotoxicity Assay

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Furin complementation. HEK293 FRT and ΔFur293 cells were stably transfected with the gene for Fur or CAT. All cell lines were then treated with the anti-transferrin receptor/PE24 RIT HB21-LR and evaluated for cytotoxicity. EC 50 (pM) values from five separate paired assays for each line are plotted. Dotted lines denote the average value for each condition and error bars indicate the standard error. Significant P -values ( P <0.05) are reported from a one-way ANOVA performed as described.

Article Snippet: Human furin cDNA (product #RC204279) was obtained from OriGene (Rockville, MD, USA) and inserted into the pcDNA5/FRT plasmid for stable transfection into HEK293 FRT cells.

Techniques: Stable Transfection, Transfection

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Cleavage assays. HEK293 FRT cells, ΔFur293 cells, and ΔFur293 cells stably expressing transgenic wild-type furin (ΔFur293/Fur) were incubated for various time intervals from 0.5 to 8 h in culture with the anti-transferrin receptor/PE24 RIT HB21-LR. Whole cell lysates were evaluated for full length and cleaved HB21-LR by western blot (panel A) and densitometry (panel B) as described. Also shown are untreated (U) cell lysates for each cell line, HB21-LR with (+) and without (−) furin treatment in vitro , and the β-actin loading control. The ratio between the furin-cleaved band intensity and the total intensity of all RIT bands at each time point is plotted in panel B. The individual densitometric analysis values (points) and mean (bar) for at least two separate assays of each cell line are shown.

Article Snippet: Human furin cDNA (product #RC204279) was obtained from OriGene (Rockville, MD, USA) and inserted into the pcDNA5/FRT plasmid for stable transfection into HEK293 FRT cells.

Techniques: Stable Transfection, Expressing, Transgenic Assay, Incubation, Western Blot, In Vitro, Control

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Complementation with mutant furin. ΔFur293 cells were stably transfected with genes for furin that contained mutations designed to impair its intracellular trafficking or catalytic function. Mutations S773A/S775A (ADA) and S773D/S775D (DDD) alter furin trafficking, while the N295A mutant (Ala-295) inhibits catalytic activity. Two separate clonal lineages stably transfected with mutant or wild-type furin were treated with the anti-transferrin receptor/PE RIT HB21-LR to assess cytotoxicity. The EC50 (pM) values from at least four separate assays for each line were normalized for furin expression levels and plotted. Dashed lines denote the average value for each clone and error bars indicate the standard error. The largest significant P -values ( P <0.05) between sets of clones from a one-way ANOVA performed as described are indicated. All P -values are reported in Table S3 .

Article Snippet: Human furin cDNA (product #RC204279) was obtained from OriGene (Rockville, MD, USA) and inserted into the pcDNA5/FRT plasmid for stable transfection into HEK293 FRT cells.

Techniques: Mutagenesis, Stable Transfection, Transfection, Activity Assay, Expressing, Clone Assay

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Cleavage by mutant furin . ΔFur293 cells stably expressing transgenic furin mutants (FurADA, FurDDD, and FurAla-295) were incubated for various time intervals from 0.5 to 8 h in culture with the anti-transferrin receptor/PE24 RIT HB21-LR. Whole cell lysates were evaluated for full length and cleaved HB21-LR by western blot (panel A) and densitometry as described. Also shown are untreated (U) cell lysates for each cell line, HB21-LR in vitro with (+) and without (−) furin treatment, and the β-actin loading control. The ratio between the furin-cleaved band intensity and the total intensity of all RIT bands at each time point is plotted in panel B. The individual densitometric analysis values (points) and mean (bar) for at least two internalization and cleavage assays in each cell line are shown.

Article Snippet: Human furin cDNA (product #RC204279) was obtained from OriGene (Rockville, MD, USA) and inserted into the pcDNA5/FRT plasmid for stable transfection into HEK293 FRT cells.

Techniques: Mutagenesis, Stable Transfection, Expressing, Transgenic Assay, Incubation, Western Blot, In Vitro, Control

Journal: Cell Reports

Article Title: TCR and Inflammatory Signals Tune Human MAIT Cells to Exert Specific Tissue Repair and Effector Functions

doi: 10.1016/j.celrep.2019.08.050

Figure Lengend Snippet:

Article Snippet: Anti-human Furin (clone 222722) AF647 , R&D Systems , Cat# IC1503R-100UG; https://www.rndsystems.com/products/human-furin-alexa-fluor-647-conjugated-antibody-222722_ic1503r.

Techniques: Virus, Recombinant, Reverse Transcription, Activation Assay, Staining, Software

Journal: Journal of Virology

Article Title: Characterization of Genotype-Specific Carboxyl-Terminal Cleavage Sites of Hepatitis B Virus e Antigen Precursor and Identification of Furin as the Candidate Enzyme

doi: 10.1128/jvi.02348-08

Figure Lengend Snippet: FIG. 8. Effects of a PC inhibitor and human furin overexpression on HBeAg processing in HEK 293, Huh7, and HepG2 cells. Cells were transfected in duplicate with HBeAg expression constructs together with an empty plasmid or a plasmid encoding human furin. One set of samples was treated with dec-RVKR-cmk. HBeAg was detected by IP-Western blot analysis. Mock, mock transfected.

Article Snippet: The human furin expression construct was obtained from OriGene (Rockville, MD).

Techniques: Over Expression, Transfection, Expressing, Construct, Plasmid Preparation, Western Blot