Journal: PLoS ONE

Article Title: A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

doi: 10.1371/journal.pone.0159868

Figure Lengend Snippet: Trypsin (A), matriptase (B), prostasin (C), factor Xa (D), neutrophil elastase (E), proteinase 3 (F), cathepsin G (G) and chymotrypsin (H) activity was assessed using their cognate fluorogenic substrates in the presence or absence of 10 μM inhibitor (QUB-F1 or furin I) (N = 3). Data represent mean ± s.e.m.; ***P<0.001.

Article Snippet: Recombinant human enzymes were sourced as follows: furin and matriptase (R&D Systems); prostasin (Sino Biological); trypsin and chymotrypsin (Sigma-Aldrich); factor Xa and neutrophil elastase (Merck Millipore) and proteinase-3 and cathepsin G (Elastin Products, Missouri).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy

doi: 10.1371/journal.pone.0194660

Figure Lengend Snippet: (A-C). Western blot detection of SDS-stable complexes between HAI-1 (H1), HAI-2 (H2), and protein nexin-1 (PN-1) and wildtype (A) , catalytically-inactive S238A (B) , and zymogen-locked R44Q (C) variants of prostasin after pre-incubation with ( A-C , lanes 2, 4, 6, and 8) or without ( A-C , lanes 1, 3, 5, and 7) recombinant human matriptase. HAI-1 and HAI-2 efficiently formed SDS-stable complexes with wildtype and catalytically-inactive prostasin after zymogen conversion ( A and B , lanes 4 and 6), whereas PN-1 only formed complex with a wildtype prostasin ( A and B , lane 8). No complexes were detected between the R44Q variant of prostasin and any of the three inhibitors ( C , lanes 4, 6, and 8). Incubation with matriptase leads to a reduction in apparent molecular weight of prostasin both before ( A , lanes 9 and 10) and after ( A , lanes 11 and 12) de-glycosylation, indicating proteolytic processing of prostasin zymogen. Positions of prostasin zymogen (black arrowhead) and activated double-chain prostasin (grey arrowhead) are indicated on the right. Location of prostasin/HAI-1 (blue asterisk), prostasin/HAI-2 (green asterisk) and prostasin/PN-1 (red asterisk) are shown directly on the blot. Positions of protein molecular weight markers is shown on the left. (D) . Western blot detection of prostasin and HAI-2 after co-immunoprecipitation from E11.5 mouse placental tissues. Placental extracts from control ( Spint2 +/+ ;Prss8 +/+ , C, lanes 1 and 4), and HAI-2-expressing ( Spint2 +/+ ;Prss8 R44Q/R44Q (Zy, lanes 2 and 5) or HAI-2-deficient ( Spint2 -/- ; Prss8 R44Q/R44Q , 0, lanes 3 and 6) prostasin zymogen-locked embryos were incubated with anti-HAI-2 (lanes 1–3) or anti-prostasin (lanes 4–6) antibody, then analyzed by Western blot using anti-prostasin (black arrowhead, top panel) or anti-HAI-2 (red arrowheads, bottom panel) antibodies. The two proteins co-immunoprecipitated in mice expressing wildtype, but not R44Q prostasin. (E) . Distribution of HAI-2 genotypes among newborn mice from Spint2 +/− ; Prss8 R44Q/+ breeding pairs. Loss of HAI-2 ( Spint2 -/- ) leads to a complete embryonic lethality in mice expressing at least one wildtype allele ( Prss8 +/+ or Prss8 R44Q/+ , collectively labeled as Prss8+ ) of prostasin ( Spint2 -/- ; Prss8+ , P<0.0001, χ 2 ) but not zymogen-locked prostasin ( Spint2 -/- ;Prss8 R44Q/R44Q ). (F) . Macroscopic appearance of newborn Spint2 -/- ;Prss8 R44Q/R44Q pups (right) and their wildtype littermate controls ( Spint2 + ;Prss8 + left). No obvious developmental abnormalities associated with the loss of HAI-2 was noticed at birth.

Article Snippet: The samples were spun at 1,000 g for 1 min to remove the beads, and the supernatant was then incubated with 3 μg goat anti-mouse HAI-2 or goat anti-mouse prostasin antibody (both R&D Systems) and 50 ul of GammaBind G Sepharose beads for 3 hours at 4 o C. The samples were spun at 1,000 g for 1 min, the supernatant was removed, and the beads were washed 3 times with 1 ml ice-cold 50 mM Tris/HCl, pH 8.0; 1% NP-40; 500 mM NaCl buffer.

Techniques: Western Blot, Incubation, Recombinant, Variant Assay, Molecular Weight, Glycoproteomics, Immunoprecipitation, Control, Expressing, Labeling

Journal: PLoS ONE

Article Title: Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy

doi: 10.1371/journal.pone.0194660

Figure Lengend Snippet: (A) . Body weight of control ( Spint2 + ;Prss8 + , n = 30), and prostasin zymogen-locked HAI-2-deficient ( Spint2 -/- ;Prss8 R44Q/R44Q , n = 13), and HAI-2-expressing ( Spint2 + ;Prss8 R44Q/R44Q , n = 47) mice at birth. HAI-2-deficient mice exhibit 20% reduction in the initial postnatal body weight (P<0.0001, Student’s t-test). (B-D) . Macroscopic appearance 4 days after birth (B) , postnatal weight gain (C) , and overall survival (D) of Spint2 -/- ;Prss8 R44Q/R44Q pups and their littermate controls. Loss of HAI-2 leads to postnatal growth retardation, inability to gain weight, and loss of viability 4–7 days after birth. (E-G”) . Macroscopic appearance of the gastrointestinal tract of control (E-G) , prostasin zymogen-locked HAI-2-deficient (E’-G’) , and HAI-2-expressing (E”-G”) mice on embryonic day (E)18.5 (E-E”) , postnatal day (P)2 (F-F”) , and postnatal day (P)4 (G-G”) . Intestines of HAI-2-deficient mice show signs of edema ( F’ , G’ , black arrows), shortening of large intestine ( F’ , G’ , left brackets) and intestinal bleeding ( G’ , black arrowhead) after birth. (H) . Relative length of large intestines (ratio of large intestine to total length of the intestines) in control, prostasin zymogen-locked HAI-2-expressing ( Spint2 + ;Prss8 R44Q/R44Q ) and HAI-2-deficient ( Spint2 -/- ;Prss8 R44Q/R44Q ) mice at E18.5, P2, and P4. The number of mice evaluated in each group and the p-values the observed differences between the controls and HAI-2-deficient mice are indicated. HAI-2-deficient mice exhibit significant shortening of the large intestine on P2 and P4. (I, I’) . H&E staining of the content of the lumen of the small (I) and large (I’) intestine of 4-days-old Spint2 -/- ;Prss8 R44Q/R44Q mice. Staining shows red blood cells ( I , arrows) and high amount of cellular material ( I , I’ , arrowheads), indicating bleeding and excessive cell shedding into the lumen. Size bars: (E-E”) 8 mm; (F-G”) 10 mm; (I, I’) 30 μm.

Article Snippet: The samples were spun at 1,000 g for 1 min to remove the beads, and the supernatant was then incubated with 3 μg goat anti-mouse HAI-2 or goat anti-mouse prostasin antibody (both R&D Systems) and 50 ul of GammaBind G Sepharose beads for 3 hours at 4 o C. The samples were spun at 1,000 g for 1 min, the supernatant was removed, and the beads were washed 3 times with 1 ml ice-cold 50 mM Tris/HCl, pH 8.0; 1% NP-40; 500 mM NaCl buffer.

Techniques: Control, Expressing, Staining

Journal: PLoS ONE

Article Title: Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy

doi: 10.1371/journal.pone.0194660

Figure Lengend Snippet: (A-C). Western blot analysis of the expression of epithelial junctional proteins, from top to bottom, EpCAM, E-cadherin, claudin -1, -2, -4, and -7, and occludin in intestines from control ( Spint2 + ;Prss8 + , lanes 1–3), HAI-2-deficient ( Spint2 −/− ;Prss8 R44Q/R44Q , lanes 4–6), and HAI-2-expressing ( Spint2 −/− ;Prss8 R44Q/R44Q , lanes 7–9) prostasin zymogen-locked mice at E18.5 ( A) , P2 (B) , and P4 (C) . Positions of the protein bands is indicated on the far right. Positions of molecular weight markers are indicated on the left. (D) . Quantification of the Western blot analysis shown in A-C . Expression in control ( Spint2 + ;Prss8 + ) tissues shown in blue, HAI-2-deficient ( Spint2 −/− ;Prss8 R44Q/R44Q ) in red, and HAI-2 expressing zymogen-locked ( Spint2 + ;Prss8 R44Q/R44Q ) in green bars. Spint2 −/− ;Prss8 R44Q/R44Q mice presented with a significantly decreased expression of EpCAM and claudin-1 as early as on E18.5, followed by a decreased expression of E-cadherin and claudin-7, and an increased expression of claudin-4 on postnatal days 2 and 4. Changes in occludin expression did not reach statistical significance, and no changes were detected in the expression of claudin-2. Data are representative of at least two independent experiments. (E). Quantification of mRNA expression of junctional proteins in intestinal tissues from P2 control (blue), HAI-2-deficient ( Spint2 −/− ;Prss8 R44Q/R44Q , red), and HAI-2 expressing zymogen-locked ( Spint2+;Prss8 R44Q/R44Q , green) mice. Only the gene encoding claudin-4 was differentially expressed, showing significant up-regulation in HAI-2-deficient tissues. D, E: P values: * <0.05; ** <0.01; *** <0.001.

Article Snippet: The samples were spun at 1,000 g for 1 min to remove the beads, and the supernatant was then incubated with 3 μg goat anti-mouse HAI-2 or goat anti-mouse prostasin antibody (both R&D Systems) and 50 ul of GammaBind G Sepharose beads for 3 hours at 4 o C. The samples were spun at 1,000 g for 1 min, the supernatant was removed, and the beads were washed 3 times with 1 ml ice-cold 50 mM Tris/HCl, pH 8.0; 1% NP-40; 500 mM NaCl buffer.

Techniques: Western Blot, Expressing, Control, Molecular Weight

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 1. Expression analysis of matriptase, HAI-1, HAI-2, and prostasin in B cancer cells by reverse- transcription/qPCR (a,b), western blotting (c–e), and flow cytometry (f). (a) Bar graph of relative mRNA expression levels of matriptase (Mat), HAI-1, HAI-2, prostasin (Pro) in Daudi (n = 4), Namalwa (n = 5), Ramos (n = 3), Raji (n = 3), JeKo-1 (n = 3), and RS4;11 cells (n = 2) using GAPDH as the reference. The prostasin bars do not appear in the bar graph, as the actual qPCR readouts were registered as “N/A” by the instrument. (b) Bar graph of mRNA quantity ratio of HAI-2 to matriptase after normalization with the GAPDH level in each cell line in (a). (c) Western blotting images of matriptase (Ab: A300-221A), HAI-2, and GAPDH. Twenty micrograms of total protein from the cell lysate of each individual culture (including 2 repeats) were analyzed. Daudi, lanes 1–3; Namalwa, lanes 4–6; Ramos, lanes 7–9. Top panel, matriptase (Mat); middle panel, HAI-2; bottom panel, GAPDH. (d) Densitometry bar graph of relative protein quantities of matriptase and HAI-2 using GAPDH as the reference. (e) The quantitative ratio of HAI-2 to matriptase in each cell line. (f) Flow cytometry histogram of matriptase expression evaluation in Ramos cells. The Ramos cells (4 × 105) were labeled with the matriptase antibody as described in the Materials and Methods section. The matriptase-positive cells are shown in the PE-A subset (blue peak). Cells without the matriptase antibody labeling (red peak) were not detected in the PE-A subset and were used as the gating control.

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Expressing, Reverse Transcription, Western Blot, Cytometry, Flow Cytometry, Labeling, Antibody Labeling, Control

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 2. Prostasin exosomes reduce matriptase quantity in B cancer cells. (a) Western blot images of matriptase (Ab: A300-221A) in samples from the cell lysate (top panel) and the conditioned media (bottom panel) after incubation with prostasin exosomes (Pro) or exosomes without prostasin (KO). The Daudi cells (2 × 105 cells each) were incubated with the exosomes in 50 µL of OPTI-MEM I/2%FBS (lanes 1–4) or RPMI medium (lanes 5–8) overnight. One-half of each cell lysate or 40 µL of each media supernatant were analyzed. (b) Western blot images of GAPDH from (a). (c) Densitometry of relative intensities of matriptase in the cell lysate or media (d). Data presented are the average intensity of lanes 1, 3, 5, 7 versus that of lanes 2, 4, 6, 8 after normalization with GAPDH in (b). (e) Western blot images of matriptase (top panel; Ab: sc-365482) in the Daudi, Namalwa, and Ramos cells treated with exosomes isolated from the HEK293T cells. Cells (2.5 × 105) were co-cultured with prostasin exosomes (Pexo, lanes 3, 6, 9) or vector exosomes (Vexo, lanes 2, 5, 8) in 100 µL of OPTI-MEM I/2%FBS. Cells without exosomes (None, lanes 1, 4, 7) were cultured in the same conditions. Bottom, GAPDH western blot image. (f) Bar graph of (e) expressed as the relative intensities of matriptase

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Western Blot, Incubation, Isolation, Cell Culture, Plasmid Preparation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 3. B cell matriptase quantity reduction by wild-type prostasin. (a) Western blot images of matriptase (Ab: sc-365482) and GAPDH in the Daudi (top two panels), Ramos (middle two panels), and Namalwa (bottom two panels) cells treated with exosomes isolated from the Calu-3 cells and sublines with over-expressed prostasin or variants. Calu-3, parent cells; KO, subline with prostasin

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Western Blot, Isolation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 5. Ectopic expression of prostasin in B cancer cells. (a) Western blot analysis of transient expression of prostasin (P) or vector alone (V) in the Daudi, Namalwa, and Ramos cells. The lysate from 2 × 105 cells of each type was analyzed. Top panel, matriptase (Ab: sc-365482); middle panel, prostasin; bottom panel, GAPDH. (b) Flow cytometry analysis of Namalwa sublines with tetracycline-induced prostasin expression or vector alone. Red peak (vector-alone cells) and sky-blue peak (prostasin-expressing cells) are samples without the prostasin antibody incubation. Orange peak (vector-alone cells) and green peak (prostasin-expressing cells) are samples incubated with the prostasin antibody. All samples were incubated with a secondary antibody conjugated with the fluorophore Cy3, and 10,000 cells of each sample were analyzed in a CytoFLEX S flow cytometer. The data were analyzed with FlowJo™software v10.8.1 and are presented in the histogram. (c) Western blot analysis of NamalwaTR sublines. One hundred thousand cells of each sample were analyzed. Lanes 1 and 4 or V, samples of the vector control subline; lanes 2 and 5 or P, samples of the subline with the wild-type prostasin; lanes 3 and 6 or M, samples of the subline with a serine active-site mutant prostasin. Left panel, cells were grown in OPTI-MEM I/2%FBS with 1 µg/mL tetracycline (with tet); right panel, cells were grown without tetracycline (no tet) for 8 days. Top two panels, matriptase antibody (sc-365482); bottom two panels, prostasin antibody. (d) Western blot analysis of tet-conditioned media from (c). Two hundred milliliters of the conditioned media were precipitated with trichloroacetic acid (TCA) (final 16.7%) at 4 ◦C overnight. The pellet was collected via centrifu- gation and analyzed. The membrane was blotted with the AF3946 human matriptase/ST14 catalytic domain antibody.

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Expressing, Western Blot, Plasmid Preparation, Flow Cytometry, Incubation, Cytometry, Software, Control, Mutagenesis, Membrane

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 6. Impact of prostasin–matriptase cascade activation on B cancer cells. (a) Bar graph of cell count for two consecutive days of B cells treated with exosomes. Namalwa, n = 7; Ramos, n = 6; Raji, n = 5; Jeko-1, n = 6. * denotes p < 0.05. (b) Growth curves of NamalwaTR-Vec and NamalwaTR-Pro cells under tetracycline induction. Left graph, cells were set at 2.5 × 105/mL on day 0 and cultured in the growth medium containing 10%FBS for 4 days. Right graph, on day 4 (reset, indicated by the arrow), the cells were diluted in OPTI-MEM I/2%FBS to 5 × 105/mL and cultured for another 5 days. Tetracycline at 1 µg/mL was added into the culture on day 0 and maintained through culturing. n = 4 for each cell line, and * denotes p < 0.05. (c) Trypsin-like serine protease activity in the conditioned media of NamalwaTR-Vec and NamalwaTR-Pro cells (n = 4). Data were analyzed in Excel with student’s t test. * denotes p < 0.05 between the two sample groups. (d) Bar graph of annexin-V-positive cells analyzed by flow cytometry. Cells under tetracycline induction were cultured for various times (week 1, n = 3; week 2, n = 4; week 3, n = 3) and subjected to direct labeling of annexin V conjugated with fluorophore allophycocyanin (APC). Ten thousand cells for each sample were analyzed on the CytoFLEX S flow cytometer. Propidium iodide staining and FSC/SSC discrimination were used for gating the live singlets, which were further analyzed for annexin V staining. (e) Bar graph of migrated cells treated with exosomes for 24 h. * denotes p < 0.05. (f) Bar graph of migrated Namalwa sublines with the induction of prostasin expression for 2–4 days and reconditioned in RPMI medium for 1 day before seeding in Transwells for migration (n = 7). * denotes p < 0.05. (g) Bar graph of invaded cells treated with exosomes for 24 h. (h) Bar graph of invaded Namalwa sublines (n = 5) treated as in (f). * denotes p < 0.05. (i) Gelatin zymography and western blot analysis. Top panel, the Ramos cells (2 × 106) in 500 µL of RPMI/0.1%BSA were treated with vector exosomes (Vexo), prostasin exosomes (Pexo), or a purified recombinant human matriptase serine protease domain (r-Mat SPD) overnight. One-fifth of the cell lysate (lanes 1–3) or 20 µL of the conditioned medium (lanes 4–6) were analyzed. The Vexo or Pexo exosomes or the r-Mat SPD alone were incubated in RPMI/0.1%BSA and used as controls (lanes 7–9). The clear bands at ~70 kDa marked by a filled arrow are matriptase. These were recognized by matriptase antibodies (middle panel). Unidentified bands with gelatinase activity marked at * locations in lanes 4, 5, 7, 8 are inherited from the exosomes, as shown in the samples with the exosomes alone (lanes 7 and 8). The band marked by the white circle is unknown. Bands at ~28 kDa marked by an unfilled arrow are r-Mat SPD. Bottom panel is GAPDH, which is detected only in the cell lysate, not in media samples or the controls without cells. (j) Gelatin zymography of B cancer cells treated as described in (i). The matriptase gelatinase activity is decreased in the cell lysate (lanes 2, 6, 10, 14) but increased in the corresponding media samples (lanes 4, 8, 12, 16) upon Pexo treatment in comparison to that of the Vexo-treated samples (in lysate, lanes 1, 5, 9, 13; in media, lanes 3, 7, 11, 15), correspondingly.

Article Snippet: Rabbit anti-human prostasin sera or pre-immune rabbit sera [7] and the matriptase antibody (A6135, ABclonal Technology, Woburn, MA, USA) were used as the primary antibodies at 1:100 dilution.

Techniques: Activation Assay, Cell Counting, Cell Culture, Activity Assay, Cytometry, Labeling, Staining, Expressing, Migration, Zymography, Western Blot, Plasmid Preparation, Recombinant, Incubation, Comparison

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Immunofluorescence analysis (extended focus image) showing uromodulin on the surface of HEK293 cells. The protein is not assembled into polymers when expressed in this cellular system. Scale bar, 21 µm. ( B ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by MDCK and HEK293 cells. HEK293 cells only secrete the longer polymerisation-incompetent uromodulin isoform (black arrowhead), while MDCK cells also secrete the shorter one (white arrowhead). ( C ) Schematic representation of the selection process employed to identify candidate enzymes for the secretion of the short uromodulin isoform. Only membrane-bound serine proteases specifically expressed by MDCK cells and by the TAL segment of the nephron, but not by HEK293 cells, were selected. ( D ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by HEK293 cells expressing wild-type or catalytically inactive human hepsin or prostasin, as indicated. Only wild-type proteases promote the secretion of the short uromodulin isoform by these cells (white arrowhead). ( E ) Immunofluorescence analysis (extended focus image) showing uromodulin on the surface of HEK293 cells expressing wild-type or catalytically inactive hepsin or prostasin, as indicated. Uromodulin polymerisation is induced only when wild-type proteases are expressed. Scale bar, 21 µm. ( F ) Representative Western blot analysis of uromodulin immunoprecipitation (upper panels) from lysates of HEK293 cells expressing hepsin or prostasin, as indicated. Both enzymes are co-immunoprecipitated when uromodulin is co-expressed in HEK293 cells (lower panels). The arrowheads point at hepsin and prostasin specific bands. DOI: http://dx.doi.org/10.7554/eLife.08887.009

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Immunofluorescence, Western Blot, Selection, Membrane, Expressing, Immunoprecipitation

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) RT-PCR analysis showing gene expression of candidate proteases hepsin ( HPN ) and prostasin ( PRSS8 ) in MDCK and HEK293 cells. Constructs containing coding sequences of the human proteases were used as PCR positive controls (C+). Expression of GAPDH is shown as a cDNA positive control. PRSS8 and HPN are exclusively expressed in MDCK cells, confirming data obtained from available transcriptomes. ( B ) Representative Western blot analysis of uromodulin, hepsin and prostasin in lysates of transfected HEK293 cells. Wild-type proteases as well as catalytically inactive enzymes were expressed in HEK293 cells, as indicated. Protein disulfide-isomerase (Pdi) is shown as a loading control. The arrowhead points at hepsin specific band. DOI: http://dx.doi.org/10.7554/eLife.08887.010

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Construct, Expressing, Positive Control, Western Blot, Transfection, Control

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Schematic representation of human uromodulin domain structure as shown in . The region not included in recombinant efUmod is shadowed. ( B ) The deletion of the elastase-sensitive fragment of uromodulin does not affect protein polymerisation on the surface of MDCK cells, as shown by immunofluorescence analysis (efUmod wt). As for full-length uromodulin , this process depends on correct protein cleavage at the physiological site, since it is abolished when the consensus cleavage site is mutated (efUmod 4Ala, carrying the mutation 586 RFRS 589 > 586 AAAA 589 ). Scale bar, 50 µm. ( C ) Purified efUmod, either wild-type (efUmod wt) or mutated at the consensus cleavage site (efUmod YAla, carrying the mutation 586 RFRS 589 > 586 AYAA 589 ), was incubated with recombinant prostasin or hepsin, as indicated. Both proteases decrease the mass of wild-type efUmod (white arrowheads in upper and middle panels) and cause the loss of its C-terminal His-tag (lower panel). Hepsin is more efficient than prostasin, as it drives complete digestion of the product, despite being used at 20x lower concentration (picomolar ratio between protease and efUmod was 1:100 for hepsin and 1:5 for prostasin, see lanes 7 and 8 for comparison). The asterisk indicates His-tagged prostasin. DOI: http://dx.doi.org/10.7554/eLife.08887.011

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Recombinant, Immunofluorescence, Mutagenesis, Purification, Incubation, Concentration Assay, Comparison

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Confocal immunofluorescence analysis showing uromodulin (green), hepsin or prostasin (red) and E-cadherin (blue) (basolateral membrane marker) in polarised MDCK cells, as indicated. Upper panels represent the reconstruction on the xz axis of merged xy scans, for which a representative image is shown in lower panels. Both serine proteases co-localise with uromodulin on the apical plasma membrane of polarised MDCK cells. z stacks = 0.3 µm. A: apical, BL: basolateral. Scale bars, 5 µm. ( B ) Transcript levels of HPN and PRSS8 , as assessed by Real-Time qPCR in MDCK cells transfected with shRNA vectors, as indicated. Expression values (normalised to glyceraldehyde-3-phosphate dehydrogenase, GAPDH) are shown as relative to cells transfected with control vector. Expression of the proteases is specifically reduced in silenced cells. Bars indicate average ± s.e.m. **p<0.01, ***p<0.001 (Student’s t test). The graph represents mean ratios of 3 independent experiments . ( C ) Immunofluorescence analysis showing uromodulin on the surface of MDCK cells transfected with control vector or with shRNA vectors targeting hepsin or prostasin, as indicated. Scale bar, 50 µm. Quantification of the average surface of uromodulin polymers shows that silencing of hepsin, but not of prostastin, substantially reduces uromodulin polymerisation on the membrane of MDCK cells. Bars indicate average ± s.e.m. ***p<0.001 (Mann-Whitney test). The graph represents mean ratios of 3 independent experiments . DOI: http://dx.doi.org/10.7554/eLife.08887.012 10.7554/eLife.08887.013 Figure 5—source data 1. Transcript level of HPN and PRSS8 in MDCK cells after shRNA transfection . DOI: http://dx.doi.org/10.7554/eLife.08887.013 10.7554/eLife.08887.014 Figure 5—source data 2. Quantification of the area of uromodulin polymers on the surface of MDCK cells after shRNA transfection . DOI: http://dx.doi.org/10.7554/eLife.08887.014

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Immunofluorescence, Membrane, Marker, Clinical Proteomics, Transfection, shRNA, Expressing, Control, Plasmid Preparation, MANN-WHITNEY

Journal: eLife

Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

doi: 10.7554/eLife.08887

Figure Lengend Snippet: ( A ) Transcript level of Prss8 , as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh ). Expression of Prss8 is detected in proximal convoluted tubules (PCT), proximal straight tubules (PST) and, to a lesser extent, in thick ascending limb (TAL) and collecting ducts (CD). Bars indicate average ± s.e.m. of 3 independent experiments . ( B ) Immunofluorescence analysis of mouse kidney sections shows strong signal of endogenous prostasin on the apical plasma membrane of proximal tubules, and weak signal on the apical plasma membrane of TAL epithelial cells where it co-localises with uromodulin. Scale bar, 20 µm. ( C ) Representative Western blot analysis of urinary uromodulin from control Prss8 lox/lox or Prss8 -/- mice. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows that uromodulin secretion is comparable between Prss8 -/- mice and control Prss8 lox/lox animals (average ± s.d., n = 5/group, ) (Student’s t test). ( D ) Representative Western blot analysis of N-deglycosylated urinary uromodulin secreted by Prss8 -/- mice or control animals. An isoform of identical molecular weight, corresponding to the short uromodulin isoform, is detected in urine samples of both genotypes (n = 5/group). ( E ) Mass spectrometry sequence coverage (55% over the entire protein) of AspN-digested mouse uromodulin (UniProt accession Q91X17) purified from urine of Prss8 -/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at F588, the same C-terminal residue identified in urinary uromodulin of wild-type mice (Santambrogio et al., 2008) and control Prss8 lox/lox animals (data not shown). ( F ) Representative MS/MS spectrum confirming the sequence of urinary uromodulin C-terminal peptide ( 573 DSTSEQCKPTCSGTRF 588 ) in Prss8 -/- mice and table of fragmented ions. DOI: http://dx.doi.org/10.7554/eLife.08887.024

Article Snippet: Human hepsin and prostasin cDNA (OriGene, Rockville, MD) were cloned in pcDNA3.1/Zeo(+) (Life Technologies).

Techniques: Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Western Blot, Control, Concentration Assay, Molecular Weight, Mass Spectrometry, Sequencing, Purification, Residue, Tandem Mass Spectroscopy

Journal: Oncotarget

Article Title: Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

doi:

Figure Lengend Snippet: A. Detection of hepsin expressed in HeLa cells. HeLa cells were transfected with full-length hepsin (WT-hepsin), an inactive hepsin catalytic mutant (S353A-hepsin), WT-hepsin and HAI-2, HAI-2, or vector alone. After 48 hours, lysates were analyzed by immunoblot and probed using anti-hepsin, anti-HAI-2, and anti-GAPDH antibodies. The 28-kDa hepsin catalytic domain, detected under reducing conditions, is a product of activation of the 51-kDa hepsin zymogen and is a measure of the presence of active hepsin. The long exposure allows detection of the low levels of active hepsin in the absence of HAI-2. The blot is representative of at least two independent experiments. B. Hepsin expression in HeLa cells enhances PrAg-PCIS toxin-induced cytotoxicity. Control and hepsin expressing HeLa cells were incubated with indicated concentrations of PrAg-PCIS and FP59 (50 ng/mL) for 6 hours. Media was then replaced and cell viability assayed after 24 hours by MTT assay. Values are the means calculated from two independent experiments performed in triplicate. * p < 0.05; ** p < 0.01. C. Detection of matriptase expressed in HeLa cells. HeLa cells were transfected with full-length matriptase (WT-matriptase), prostasin, vector alone, or were co-transfected with matriptase, prostasin, and HAI-1. After 48 hours, lysates were analyzed by immunoblot using anti-matriptase, anti-prostasin, anti-HAI-1, and anti-GAPDH antibodies. The 28-kDa matriptase catalytic domain detected under reducing conditions is evidence of active matriptase produced upon activation of the 70-kDa zymogen form of matriptase. The blot is representative of at least two independent experiments. D. Matriptase expression in HeLa cells does not enhance PrAg-PCIS toxin-induced cytotoxicity. Control and matriptase expressing HeLa cells were incubated with indicated concentrations of PrAg-PCIS and FP59 (50 ng/mL) for 6 hours. Media was then replaced and cell viability assayed after 24 hours by MTT assay. Values are the means calculated from two independent experiments performed in triplicate.

Article Snippet: Recombinant mouse testisin (6820-SE-10), human hepsin (4776-SE-10), human prostasin (4599-SE), and HAT (2695-SE) were purchased from R&D Systems.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Activation Assay, Expressing, Control, Incubation, MTT Assay, Produced

Journal: Heliyon

Article Title: Chymase uptake by cardiomyocytes results in myosin degradation in cardiac volume overload

doi: 10.1016/j.heliyon.2019.e01397

Figure Lengend Snippet: RBL-2H3 mast cells immediately after addition of ionomycin (5 μg/ml) (middle panel) and 10 minutes after degranulation. Notice membrane projections (white arrow) and released granules and debris (black arrows). MS analysis was performed on whole cell lysates and supernatant after degranulation. Coomassie gel of degranulated (DG) RBL-2H3 demonstrates Lane 1 conditioned media control, Lane 2 conditioned media after DG, Lane 3 is control cell lysate, Lane 4 is cell lysate after DG. Arrows indicate a 27 kD band and a 43 kD band which Mass spectroscopy analysis identified as rMCP-2 and actin, respectively. Mass spectroscopy analyses were done through the UAB Mass Spectroscopy Core Facility.

Article Snippet: Recombinant rat mast cell protease 1(rMCP-1; 35 ng Cusabio Technology LLC #CSB-EP357723RA), 2(rMCP-2; 35 ng, Cusabio Technology LLC #CSB-EP360441RA), 4(rMCP-4; 35 ng, Cusabio Technology LLC #CSB-EP311424RA), purified human skin chymase (50 ng, Enzo Life Sciences Inc. #BML-SE281-0000) or rat LV tissue homogenates (2.5 ug) were denatured at 95 °C for 5 minutes in NuPAGETM LDS Sample Buffer containing NuPAGETM sample reducing agent (Invitrogen).

Techniques: Membrane, Control, Mass Spectrometry

Journal: Heliyon

Article Title: Chymase uptake by cardiomyocytes results in myosin degradation in cardiac volume overload

doi: 10.1016/j.heliyon.2019.e01397

Figure Lengend Snippet: Western blot showing specificity of custom developed antibodies to Rat MCP-2. The custom 3F4.5 mouse monoclonal and the rabbit polyclonal antibody recognize the intact recombinant rat mast cell protease-2 (rMCP-2) protein (A) and do not cross react with rMCP-1 (B), rMCP-4 (C) or purified human chymase. The recombinant rMCP-1, 2 and 4 have a C or N-terminal Myc tag and the left panel illustrates that the recombinant proteins run at ∼27 kD. The 3F4.5 monoclonal and the rabbit polyclonal antibodies recognize the rMCP-2 protein (A – 2 nd and 3 rd panels) but not purified rMCP-1 (B), rMCP-4 (C) or human chymase (hChymase; 2 nd and 3 rd panels in each row). The R&D mouse monoclonal recognizes CMA1, the purified human chymase, but not the rMCP-1, 2 or 4 proteins (4 th panel). As depicted in the far right panel 3F4.5 specifically recognizes rat MCP-2.

Article Snippet: Recombinant rat mast cell protease 1(rMCP-1; 35 ng Cusabio Technology LLC #CSB-EP357723RA), 2(rMCP-2; 35 ng, Cusabio Technology LLC #CSB-EP360441RA), 4(rMCP-4; 35 ng, Cusabio Technology LLC #CSB-EP311424RA), purified human skin chymase (50 ng, Enzo Life Sciences Inc. #BML-SE281-0000) or rat LV tissue homogenates (2.5 ug) were denatured at 95 °C for 5 minutes in NuPAGETM LDS Sample Buffer containing NuPAGETM sample reducing agent (Invitrogen).

Techniques: Western Blot, Recombinant, Purification