sheep polyclonal anti matriptase catalytic domain antibody  (R&D Systems)

Journal: FEBS letters

Article Title: Proteolytic cleavage of Trop2 at Arg87 is mediated by matriptase and regulated by Val194.

doi: 10.1002/1873-3468.13899

Figure Lengend Snippet: Fig. 2. Identification of Trop2 as a target for matriptase. (A) Sequence alignment of a part of human EpCAM and human Trop2 protein sequences. Scissors denote reported N-terminal cleavage site in human EpCAM between Arg80 and Arg81 [19]. It corresponds to Arg87 and Thr88 (boxed) in human Trop2 which were selected for generating alanine substitution mutants. (The conserved residues between the two proteins are indicated by asterisks below the sequence.) Amino acid numbering is including the signal peptide. (B) Expression of WT Trop2-HA and the indicated alanine substitution mutants. Total cell lysate from HEK293 cells transiently transfected with either WT Trop2- HA or R87A Trop2-HA or T88A Trop2-HA was immunoblotted before (left) and after PNGase treatment (right) using anti-HA antibody (upper panel) and anti-GAPDH antibody (lower panel) (C) Cell surface localization of Trop2 (green) in nonpermeabilized HEK293 cells transiently expressing the indicated Trop2-HA constructs visualized by indirect immunofluorescence using anti-Trop2 ECD antibody. DAPI (blue) was used as a nuclear stain. Bar = 10 µm. HEK293 cells transfected with an EV served as a negative control. (D) HEK293 cells were cotransfected with the indicated amount of WT Trop2-HA and either WT matriptase (Mat-HA) or inactive matriptase (Mat-M-HA) constructs. Cell lysates were prepared 48 h post-transfection followed by immunoblotting with anti-HA antibody to detect matriptase as well as Trop2 protein. GAPDH is the loading control (lower panel). Filled triangle and hollow triangle represents full-length Trop2 and DN-Trop2, respectively. (E) Immunoblot showing matriptase expression in the total cell lysates of the indicated cell lines using anti-matriptase antibody (upper panel). Expression of GAPDH in each cell line serves as the loading control (lower panel).

Article Snippet: Antibodies used in this study include a goat polyclonal anti-Trop2 antibody directed against ECD of Trop2 (1 : 3000, AF650; R & D Systems, Minneapolis, MN, USA), rabbit monoclonal antibody targeted against C-terminal region of Trop2 (1 : 2000, ab214 488; abcam, Cambridge, MA, USA), mouse monoclonal anti-HA (1 : 1000, ab18 181; abcam), anti-CD63 (1 : 500, ab59 479; abcam), sheep polyclonal anti-matriptase catalytic domain antibody (1 : 1000, AF3946; R & D Systems), mouse monoclonal antibody against GAPDH (1 : 10 000, MA5-15738; Thermo Fisher Scientific), and mouse monoclonal anti-Flag antibody (1 : 1000, F1804; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Sequencing, Expressing, Transfection, Construct, Staining, Negative Control, Western Blot, Control

Journal: FEBS letters

Article Title: Proteolytic cleavage of Trop2 at Arg87 is mediated by matriptase and regulated by Val194.

doi: 10.1002/1873-3468.13899

Figure Lengend Snippet: Fig. 3. Homology modeling of Trop2 ECD dimer. (A) Modeled structure of Trop2 ECD (1–275) dimer. Monomeric subunits are color coded (cyan and golden yellow). Positions of matriptase cleavage site between Arg87 and Thr88 and the predicted ADAM17 cleavage site Val194 is highlighted as red sticks for one monomer and as indigo sticks for another monomer. Zoomed-in image shows that the Arg87 and Thr88 of one monomer are in the same plane as that of Val194 of another monomer. (B) Sequence alignment of a part of mouse and human Trop2 protein sequences. Val188, which is a reported ADAM17 cleavage site in mTrop2, is denoted by scissors [16]. It corresponds to Val194 (boxed) in human Trop2. The conserved residues are indicated by asterisks below the sequence. Red arrows denote the amino acids (Lys189, Val194, and His195) selected for site-directed mutagenesis.

Article Snippet: Antibodies used in this study include a goat polyclonal anti-Trop2 antibody directed against ECD of Trop2 (1 : 3000, AF650; R & D Systems, Minneapolis, MN, USA), rabbit monoclonal antibody targeted against C-terminal region of Trop2 (1 : 2000, ab214 488; abcam, Cambridge, MA, USA), mouse monoclonal anti-HA (1 : 1000, ab18 181; abcam), anti-CD63 (1 : 500, ab59 479; abcam), sheep polyclonal anti-matriptase catalytic domain antibody (1 : 1000, AF3946; R & D Systems), mouse monoclonal antibody against GAPDH (1 : 10 000, MA5-15738; Thermo Fisher Scientific), and mouse monoclonal anti-Flag antibody (1 : 1000, F1804; Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Sequencing, Mutagenesis

Journal: Human molecular genetics

Article Title: SPINT2 (HAI-2) missense variants identified in congenital sodium diarrhea/tufting enteropathy affect the ability of HAI-2 to inhibit prostasin but not matriptase.

doi: 10.1093/hmg/ddy394

Figure Lengend Snippet: Figure 2. HAI-2 SCSD-associated variants Phe161Val, Tyr163Cys and Gly168Ser stabilize matriptase and display unaffected inhibitory effect toward matriptase. (A) Each column represents the rate of turnover of the chromogenic substrate S-2288 without antibody (PBS, light grey column), with antibody aZ-mAb-6 inhibiting matriptase activity (aZ-mAb-6, black column) or with a control antibody (control, dark grey column) in extracts obtained by lysis of HEK293 cells transiently transfected

Article Snippet: All samples for WB were boiled under reducing conditions and blots were probed with primary polyclonal sheep anti-human matriptase antibody (cat. no. AF3946, R&D systems, Abington, United Kingdom) (1:1000), polyclonal rabbit anti-human HAI2 antibody (cat. no. HPA011101, Sigma) (1:1000), monoclonal mouse anti-human prostasin (cat. no. 612173, BD Transduction LaboratoriesTM, Albertslund, Denmark) recognizing prostasin and with GAPDH antibody (cat. no. ZG003, Thermo Fischer Scientific, Hvidovre, Denmark) (1:2000) as control.

Techniques: Activity Assay, Control, Lysis, Transfection

Journal: Human molecular genetics

Article Title: SPINT2 (HAI-2) missense variants identified in congenital sodium diarrhea/tufting enteropathy affect the ability of HAI-2 to inhibit prostasin but not matriptase.

doi: 10.1093/hmg/ddy394

Figure Lengend Snippet: Figure 3. HAI-2 SCSD-associated variants Phe161Val, Tyr163Cys and Gly168Ser display reduced ability to inhibit prostasin-catalyzed cleavage. Extracts obtained by lysis of HEK293 cells transiently transfected with matriptase Ser805Ala (mat Ser805Ala) alone, matriptase Ser805Ala with prostasin (pro), matriptase Ser805Ala and prostasin together with HAI-2 WT or mutated HAI-2 variants (Cys47Phe/Arg48Leu, Arg143Leu, Phe161Val, Tyr163Cys or Gly168Ser), as indicated above the figure,

Article Snippet: All samples for WB were boiled under reducing conditions and blots were probed with primary polyclonal sheep anti-human matriptase antibody (cat. no. AF3946, R&D systems, Abington, United Kingdom) (1:1000), polyclonal rabbit anti-human HAI2 antibody (cat. no. HPA011101, Sigma) (1:1000), monoclonal mouse anti-human prostasin (cat. no. 612173, BD Transduction LaboratoriesTM, Albertslund, Denmark) recognizing prostasin and with GAPDH antibody (cat. no. ZG003, Thermo Fischer Scientific, Hvidovre, Denmark) (1:2000) as control.

Techniques: Lysis, Transfection

Journal: Human molecular genetics

Article Title: SPINT2 (HAI-2) missense variants identified in congenital sodium diarrhea/tufting enteropathy affect the ability of HAI-2 to inhibit prostasin but not matriptase.

doi: 10.1093/hmg/ddy394

Figure Lengend Snippet: Figure 5. Proposed schematic structure of inhibitory complexes between prostasin or matriptase and HAI-2 WT or SCSD-mutated HAI-2. A schematic overview of suggested HAI-2 inhibitory complexes between (A) prostasin (green) and WT HAI-2, (B) prostasin and SCSD-associated HAI-2 mutants, (C) matriptase (red) and WT HAI-2

Article Snippet: All samples for WB were boiled under reducing conditions and blots were probed with primary polyclonal sheep anti-human matriptase antibody (cat. no. AF3946, R&D systems, Abington, United Kingdom) (1:1000), polyclonal rabbit anti-human HAI2 antibody (cat. no. HPA011101, Sigma) (1:1000), monoclonal mouse anti-human prostasin (cat. no. 612173, BD Transduction LaboratoriesTM, Albertslund, Denmark) recognizing prostasin and with GAPDH antibody (cat. no. ZG003, Thermo Fischer Scientific, Hvidovre, Denmark) (1:2000) as control.

Techniques:

Journal: PLoS genetics

Article Title: Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

doi: 10.1371/journal.pgen.1004470

Figure Lengend Snippet: Figure 1. Combined loss of matriptase/prostasin- and PAR-2-dependent proteolytic pathways leads to embryonic lethality. (A). Matriptase haploinsufficiency decreases survival of PAR-2-deficient mice. Genotype distribution among 706 pre-weaning offspring from interbred F2rl1+/26F2rl1+/2;St14+/2 mice. A normal distribution of F2rl1 alleles was observed in St14+/+ background, whereas the number of F2rl12/2 mice heterozygous for matriptase was significantly decreased (P,0.0001). (B). Genotype distribution among 272 newborn offspring from interbred F2rl1+/2;St14+/26F2rl1+/2;St14+/2 mice. F2rl1 alleles were found in the expected Mendelian ratio in St14+/+ mice, whereas numbers of F2rl12/2;St14+/2

Article Snippet: 80 mg of total protein was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human matriptase (R&D Systems), mouse anti-cow desmoglein 1 and 2 (Fitzgerald Industries International, Acton, MA), mouse anti-human Gcm1 (Abcam, Cambridge, MA), rabbit anti-human syncytinA (SantaCruz Biotechnology), rabbit anti-human pan-cadherin, rabbit anti-human GAPDH (both Cell Signaling Technology, Danvers, MA), rabbit anti-human claudin1, and mouse anti-human claudin-2 (both Invitrogen, Carlsbad, CA) primary antibodies, and goat anti-mouse, mouse anti-rabbit (both DakoCytomation), or donkey anti-sheep (Sigma-Aldrich) secondary antibodies conjugated to alkaline phosphatase.

Techniques:

Journal: PLoS genetics

Article Title: Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

doi: 10.1371/journal.pgen.1004470

Figure Lengend Snippet: Figure 2. Loss of PAR-2 and matriptase does not affect development of the embryo proper. (A–C). Evaluation of the expression pattern of St14 (A), Prss8 (B), and F2rl1 (C) genes in E14.5 mouse embryo by in situ hybridization obtained from the Eurexpress digital transcriptome atlas. All three genes show highly overlapping pattern of expression in the epithelia of developing skin (sk), oral (oc) and nasal (nc) cavities, salivary gland (sg), lungs (l), kidney (k), and gut (g). Matriptase and prostasin, but not PAR-2, were also detected in the developing structures of the inner ear (ie). (D). Macroscopic appearance of F2rl12/2;St142/2, and wild-type littermate control embryos at E13.5. No obvious developmental abnormality was observed in the PAR-2 and matriptase double-deficient animals. (E) Total body weight of E13.5 offspring from F2rl1+/2;St14+/2 breeding pairs. None of the embryos with a decreased combined gene dosage of F2rl1 and St14 genes showed any signs of growth retardation. (G–I9). H&E staining of embryonic tissues with the highest relative expression of PAR-2 and matriptase. A comparative histological analysis of liver (F, F9), lungs (G, G9), kidneys (H, H9), and intestines (I, I9) from the control (F, G, H, and I) and littermate F2rl12/2;St142/2 (F9, G9, H9, and I9) E13.5 embryos indicates normal development of embryonic tissues in the absence of PAR-2 and matriptase function. Scale bars: (A–C) 1 mm, (F–I9) 100 um. doi:10.1371/journal.pgen.1004470.g002

Article Snippet: 80 mg of total protein was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human matriptase (R&D Systems), mouse anti-cow desmoglein 1 and 2 (Fitzgerald Industries International, Acton, MA), mouse anti-human Gcm1 (Abcam, Cambridge, MA), rabbit anti-human syncytinA (SantaCruz Biotechnology), rabbit anti-human pan-cadherin, rabbit anti-human GAPDH (both Cell Signaling Technology, Danvers, MA), rabbit anti-human claudin1, and mouse anti-human claudin-2 (both Invitrogen, Carlsbad, CA) primary antibodies, and goat anti-mouse, mouse anti-rabbit (both DakoCytomation), or donkey anti-sheep (Sigma-Aldrich) secondary antibodies conjugated to alkaline phosphatase.

Techniques: Expressing, In Situ Hybridization, Control, Staining

Journal: PLoS genetics

Article Title: Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

doi: 10.1371/journal.pgen.1004470

Figure Lengend Snippet: Figure 3. A combined loss of PAR-2 and matriptase leads to underdevelopment of placental labyrinth. (A, B). Immunohistochemical detection of matriptase (A) and prostasin (B) in mouse placenta at E12.5. Expression of both proteins was observed in chorionic epithelium (A and B, arrowheads) and in the differentiated syncytiothrophoblast layer of the labyrinth (A and B, arrows). Prostasin, but not matriptase, was also detected in the mononuclear cytotrophoblast cells within the labyrinth (B, open arrowheads). (C). Histological analysis of PAR-2 expression in the developing placenta from E12.5 F2rl1-bgal knock-in embryos. Beta-galactosidase reporter activity was detected both in chorionic epithelium (arrowheads) and in the syncytiothrophoblast layer surrounding fetal vessels within the labyrinth (arrows). (D, E). Histological evaluation of the placental development in control (D) and F2rl12/2;St142/2 (E) animals at E13.5. H&E staining confirmed the presence of all major structural components of the mouse placenta, including allantoic mesenchyme (al), placental labyrinth (lb), spongiotrophoblast layer (sp, examples with open arrowheads), and trophoblast giant cells (tgc, examples with arrowheads), in the double-deficient embryos (F–I). Immunohistochemical visualization of fetal vasculature in the E13.5 placentas. Low (F, H) and high (G, I) magnification images of the staining for the endothelial cell marker PECAM-1/CD31 in the placentas of the control (F, G) and F2rl12/2;St142/2 (H,I) mice shows presence of the branched fetal vascular tree (F–I, arrows) within the placental labyrinth of the PAR-2/matriptase double-deficient animals, although the apparent vascular density is lower than in the wildtype littermate control tissues. (J, K). Quantification of the thickness of the placental labyrinth (J) and the number of fetal vessels (K) within the labyrinth of control (F2rl1+;St14+) and F2rl12/2;St142/2 E12.5 and E13.5 placentas. The measurements show decreased thickness and vascularization of the labyrinth layer in F2rl12/2;St142/2

Article Snippet: 80 mg of total protein was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human matriptase (R&D Systems), mouse anti-cow desmoglein 1 and 2 (Fitzgerald Industries International, Acton, MA), mouse anti-human Gcm1 (Abcam, Cambridge, MA), rabbit anti-human syncytinA (SantaCruz Biotechnology), rabbit anti-human pan-cadherin, rabbit anti-human GAPDH (both Cell Signaling Technology, Danvers, MA), rabbit anti-human claudin1, and mouse anti-human claudin-2 (both Invitrogen, Carlsbad, CA) primary antibodies, and goat anti-mouse, mouse anti-rabbit (both DakoCytomation), or donkey anti-sheep (Sigma-Aldrich) secondary antibodies conjugated to alkaline phosphatase.

Techniques: Immunohistochemical staining, Expressing, Knock-In, Activity Assay, Control, Staining, Marker

Journal: PLoS genetics

Article Title: Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

doi: 10.1371/journal.pgen.1004470

Figure Lengend Snippet: Figure 4. Placental expression of matriptase restores survival in PAR-2 and matriptase double-deficient embryos. (A). Schematic depiction of the strategy used to assess contribution of placental matriptase to the survival of the F2rl12/2;St142/2 embryos. Meox2-Cre allele that drives the expression of Cre recombinase to the embryonic but not placental tissues was introduced into the mice carrying one null and one conditional allele of matriptase gene (St142/fl). The resulting mice retain expression of matriptase in the placenta but not in any of the embryonic tissues. Both the embryo and the placenta are also PAR-2-deficient (F2rl12/2). (B). Western blot analysis of matriptase expression in the embryo (top) and the placenta (bottom) of the E13.5 Meox2-Cre;St142/fl (lanes 1–3), and their St14+/fl (lanes 4–6) and St142/2 (lane 7) littermate control animals. Both the embryos and the placentas of the control mice showed detectable levels of matriptase protein (arrowheads on the right), whereas only placental tissue retained matriptase expression in the Meox2-Cre;St142/fl embryos. No matriptase expression was detected in either embryos or placentas of animals carrying two knockout alleles of matriptase (St142/2). GAPDH signal is shown to indicate equal loading. Positions of molecular weight markers (kDa) are shown on left. (C). Allele distribution of the F2rl1 gene among newborn matriptase-deficient offspring from interbred F2rl1+/2; St14+/26F2rl1+/2;St14+/2 (St142/2, left panels) or Meox2-Cre;F2rl1+/2;St14+/26F2rl1+/2;St142/fl (Meox2-Cre;St142/fl, right panels) breeder mice. Placental expression of matriptase restores embryonic survival of the PAR-2 and matriptase double-deficient mice. (D, E). Macroscopic appearance of the head of control (D) or Meox2-Cre;F2rl12/2;St142/fl (E) mice at birth. The Meox2-Cre;F2rl12/2;St142/fl newborns reproduce phenotypes of mice with complete matriptase deficiency, including the lack of whiskers. (F). Western blot analysis of matriptase expression in two Meox2-Cre;F2rl12/2;St142/fl (lanes 1–2) and two littermate control (lanes 3–4) newborn mice. No residual expression of matriptase protein (arrowhead on the right) was detected in the Meox2- Cre;F2rl12/2;St142/fl mice. Positions of molecular weight markers (kDa) are shown on left. doi:10.1371/journal.pgen.1004470.g004

Article Snippet: 80 mg of total protein was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human matriptase (R&D Systems), mouse anti-cow desmoglein 1 and 2 (Fitzgerald Industries International, Acton, MA), mouse anti-human Gcm1 (Abcam, Cambridge, MA), rabbit anti-human syncytinA (SantaCruz Biotechnology), rabbit anti-human pan-cadherin, rabbit anti-human GAPDH (both Cell Signaling Technology, Danvers, MA), rabbit anti-human claudin1, and mouse anti-human claudin-2 (both Invitrogen, Carlsbad, CA) primary antibodies, and goat anti-mouse, mouse anti-rabbit (both DakoCytomation), or donkey anti-sheep (Sigma-Aldrich) secondary antibodies conjugated to alkaline phosphatase.

Techniques: Expressing, Western Blot, Control, Knock-Out, Molecular Weight

Journal: PLoS genetics

Article Title: Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

doi: 10.1371/journal.pgen.1004470

Figure Lengend Snippet: Figure 5. Loss of matriptase and PAR-2 function impairs formation of the feto-maternal barrier. (A). Relative uptake of the transcellular transport marker 3-methyl-D-glucose injected into the maternal bloodstream by E12.5–13.5 embryos. Placentas of matriptase (F2rl1+/+,St142/2) or PAR-2 (F2rl12/2;St14+/+) single-deficient embryos supported glucose transport at a rate comparable to the wildtype littermate controls (F2rl1+;St14+), whereas matriptase and PAR-2 double-deficient mice (F2rl12/2;St142/2) showed about 20% decrease in the glucose uptake (P,0.01). (B). Relative uptake of the paracellular transport marker carboxy-inulin injected into the maternal bloodstream by E12.5–13.5 embryos. Diffusion of carboxy-inulin across the placental epithelium was strongly dependent on gene dosage of both matriptase and PAR-2, showing 247% and 145%, respectively, increase in St142/2 and F2rl12/2 single-deficient embryos, and 402% and 351% increase, respectively, in F2rl1+/2;St142/2 and F2rl12/2;St14+/2

Article Snippet: 80 mg of total protein was loaded on 4–12% reducing SDS-PAGE and analyzed by Western blotting using a polyclonal sheep anti-human matriptase (R&D Systems), mouse anti-cow desmoglein 1 and 2 (Fitzgerald Industries International, Acton, MA), mouse anti-human Gcm1 (Abcam, Cambridge, MA), rabbit anti-human syncytinA (SantaCruz Biotechnology), rabbit anti-human pan-cadherin, rabbit anti-human GAPDH (both Cell Signaling Technology, Danvers, MA), rabbit anti-human claudin1, and mouse anti-human claudin-2 (both Invitrogen, Carlsbad, CA) primary antibodies, and goat anti-mouse, mouse anti-rabbit (both DakoCytomation), or donkey anti-sheep (Sigma-Aldrich) secondary antibodies conjugated to alkaline phosphatase.

Techniques: Marker, Injection, Diffusion-based Assay