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

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

doi: 10.1074/jbc.m114.574400

Figure Lengend Snippet: FIGURE 1. Specific detection of endogenous matriptase in intestinal epithelium. The distribution of E-cadherin (A, D, G, J) and matriptase (B, E, H, and K) in small intestine (A–F) and large intestine (G–L) of matriptase-sufficient (Villin-Cre/0; St14fl/) (A–C, G–I) and intestinal matriptase-deficient (Villin-Cre/0; St14fl/) mice (D–F, J–L) demonstrating that the antibody AF3946 is matriptase specific. Matriptase and E-cadherin are merged in C, F, I, and L. Scale bar, 20 m. The data are representative of two similar experiments.

Article Snippet: Before applying the goat anti-mouse HAI-2 antibody to the PVDF membrane for overnight probing, the antibody was pre-absorbed with 10 g of protein from HAI-2 knock-out lysate for 30 min at 4 °C in 1% nonfat dry milk/PBS-T. Antibodies used for Caco-2 cell lysates were sheep anti-human matriptase (AF3946, R&D Systems), mouse anti-human matriptase monoclonal antibody M69 (39), goat anti-human HAI-1 (AF1048, R&D Systems), polyclonal rabbit anti-human SPINT2 (HPA011101, Sigma) and mouse anti-human prostasin (catalogue no. 612173, BD Transduction Laboratories).

Techniques:

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

doi: 10.1074/jbc.m114.574400

Figure Lengend Snippet: FIGURE2.LossofHAI-1doesnotaffecttheabundanceorsubcellulardistributionofmatriptaseinepitheliumofthelargeandsmallintestine.A,lysates of intestines from 6-week-old HAI-1-ablated mice (Spint1fl/; Cre, lanes 1–3) and littermate controls expressing HAI-1 (Spint1fl/ or Spint1fl/; Cre, lanes 4 and 5). Matriptase-deficient intestine (Villin-Cre/0; St14fl/) was included as a control for antibody specificity in lane 6. The lysates were boiled, reduced, and analyzed by Western blot using antibodies against HAI-1 (top panel), matriptase (second panel from top), and HAI-2 (second panel from bottom). Tubulin was used as a loading control (bottom panel). The specific proteins are indicated on the right, and molecular markers are indicated on the left (kDa). The data are representative of three similar experiments. The distribution of matriptase (C, G, K, and O) and E-cadherin (B, F, J, N) in small intestine (B–I) and large intestine (J–Q) in mice expressing HAI-1 (B–E and J–M) and HAI-1-depleted mice (F–I and N–Q). Matriptase and E-cadherin are merged in D, H, L, and P with enlargements ofindicatedarea(shownwithwhitedottedline)inE,I,M,andQ,respectively.HAI-1stainingofthesametissueisrepresentedintheinsetinC,G,KandOtogether with E-cadherin (inset, B, F, J, and N). The merge of HAI-1 and E-cadherin is shown in the inset in D, H, L, and P. Scale bar in B, 20 m, representative for B–D, F–H, J–L, N–P. Scale bar in E, 5 m, representative for E, I, M, and Q. The data are representative of three similar experiments.

Article Snippet: Before applying the goat anti-mouse HAI-2 antibody to the PVDF membrane for overnight probing, the antibody was pre-absorbed with 10 g of protein from HAI-2 knock-out lysate for 30 min at 4 °C in 1% nonfat dry milk/PBS-T. Antibodies used for Caco-2 cell lysates were sheep anti-human matriptase (AF3946, R&D Systems), mouse anti-human matriptase monoclonal antibody M69 (39), goat anti-human HAI-1 (AF1048, R&D Systems), polyclonal rabbit anti-human SPINT2 (HPA011101, Sigma) and mouse anti-human prostasin (catalogue no. 612173, BD Transduction Laboratories).

Techniques: Expressing, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

doi: 10.1074/jbc.m114.574400

Figure Lengend Snippet: FIGURE 3. Normal levels and distribution of matriptase in the intestine of HAI-1 knock-out mice in the prostasin hypomorphic background fr/fr. A, lysates of intestines from 1-year-old HAI-1 knock-out mice (Spint1/; Prss8fr/fr, lanes 1–3) and littermate controls expressing HAI-1 (Spint1/ or Spint1/; Prss8fr/fr, lanes 4–6). Matriptase-deficient (Villin-Cre/0, St14fl/) intestine was included as control (lane 7). The lysates were boiled, reduced, and analyzed by Western blotting using antibodies against HAI-1 (top panel), matriptase (second panel from top), HAI-2 (third panel from top) and prostasin (second panel from bottom). Tubulin was used as loading control (bottom panel). The specific proteins are indicated on the right and molecular markers are indicated on the left (kDa).Thedataarerepresentativeofthreesimilarexperiments.Thedistributionofmatriptase(C,G,K,andO)andE-cadherin(B,F,J,andN)insmallintestine(B–I) and large intestine (J–Q) in mice expressing HAI-1 (B–E and J–M) and HAI-1-deficient mice (F–I and N–Q). Matriptase and E-cadherin is merged in D, H, L, and P with enlargements of the indicated area (shown with white dotted line) in E, I, M, and Q, respectively. Scale bar in B, 20 m, representative for B–D, F–H, J–L, N–P. Scale bar in E, 5 m, representative for E, I, M, and Q. The data are representative of two similar experiments.

Article Snippet: Before applying the goat anti-mouse HAI-2 antibody to the PVDF membrane for overnight probing, the antibody was pre-absorbed with 10 g of protein from HAI-2 knock-out lysate for 30 min at 4 °C in 1% nonfat dry milk/PBS-T. Antibodies used for Caco-2 cell lysates were sheep anti-human matriptase (AF3946, R&D Systems), mouse anti-human matriptase monoclonal antibody M69 (39), goat anti-human HAI-1 (AF1048, R&D Systems), polyclonal rabbit anti-human SPINT2 (HPA011101, Sigma) and mouse anti-human prostasin (catalogue no. 612173, BD Transduction Laboratories).

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

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

doi: 10.1074/jbc.m114.574400

Figure Lengend Snippet: FIGURE 4. Reduced expression of matriptase in HAI-2-deficient intestinal epithelium. A, lysates of intestines from one-day-old HAI-2 expressing mice (Spint2/; Prss8fr/fr, lanes 1 and 2) and HAI-2-deficient littermates (Spint2/;Prss8fr/fr, lanes 3 and 4). Matriptase knock-out intestine (St14/) from newborn mice was included as control (lane 5). The lysates were boiled, reduced and analyzed by SDS-PAGE and Western blot using antibodies against HAI-2 (top panel), matriptase(secondpanelfromtop),HAI-1(thirdpanelfromtop),andprostasin(secondpanelfrombottom).Thehighermolecularformsofprostasininmatriptase knock-out tissue is marked with a black arrowhead. Tubulin was used as loading control (bottom panel). The specific proteins are indicated on the right, and molecular markers are indicated on the left (kDa). The data are representative of three similar experiments. B, mRNA was isolated from intestine from newborn Spint2/(n 9), Spint2 / (n 8), and Spint2/ (n 4) mice, all in a prostasin hypomorphic background (Prss8fr/fr). St14 mRNA levels were analyzed by real-time PCR and are shown relative to 18 S ribosomal RNA. The distribution of matriptase (D, H, L, and P) and E-cadherin (C, G, K, and O) in small intestine (C–J) and large intestine (K-R) in mice expressing HAI-2 (C–F and K–N) and HAI-2-deficient mice (G–J and O–R). Unspecific matriptase staining, possibly from residual maternal milk in the intestine, (marked with a white arrowhead in D) is also observed in matriptase-deficient mice (inset in 4D, indicated with white arrowhead). Matriptase and E-cadherin are merged in E, I, M, and Q and enlargements of the area indicated with the white dotted line are shown in F, J, N, and R, respectively. Scale bar in C 20 m, is representative for C–E, G–I, K—M, and O–Q. Scale bar in F 5 m, is representative for F, J, N, and R. The data are representative of three similar experiments.

Article Snippet: Before applying the goat anti-mouse HAI-2 antibody to the PVDF membrane for overnight probing, the antibody was pre-absorbed with 10 g of protein from HAI-2 knock-out lysate for 30 min at 4 °C in 1% nonfat dry milk/PBS-T. Antibodies used for Caco-2 cell lysates were sheep anti-human matriptase (AF3946, R&D Systems), mouse anti-human matriptase monoclonal antibody M69 (39), goat anti-human HAI-1 (AF1048, R&D Systems), polyclonal rabbit anti-human SPINT2 (HPA011101, Sigma) and mouse anti-human prostasin (catalogue no. 612173, BD Transduction Laboratories).

Techniques: Expressing, Knock-Out, Control, SDS Page, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Staining

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

doi: 10.1074/jbc.m114.574400

Figure Lengend Snippet: FIGURE 5. Reduction of HAI-2 levels enhances matriptase activation and cell surface shedding in intestinal epithelial cell monolayers. A, schematic depiction of full-length matriptase (top, left), SEA domain-cleaved matriptase (left, middle), activation site-cleaved matriptase (left, bottom), activated matrip- tase in complex with HAI-1 (right, top), and two forms of the matriptase-HAI-1 complex once shed from the cell surface (right, middle, and bottom). Positions of the SEA domain cleavage (Gly149) and the activation cleavage site (Arg614) are shown with arrowheads. The binding site for the antibody used to identify full-length, SEA domain-cleaved and activated matriptase by Western blot after reducing SDS-PAGE is indicated with a schematic drawing of an antibody (left panel). Also, the binding site for the antibody used to identify matriptase-HAI-1 complexes in the cell or as shed forms by Western blot under non- boiling/non-reducing conditions is indicated (right panel). B and C, three HAI-2-silencing siRNAs (siSPINT2 a–c, lanes 1–4) and three scrambled controls (siCTRL a–c, lanes 5–8) were transfected into Caco-2 cells. Three days post-transfection, the cell lysates (B) were analyzed by Western blot using antibodies against HAI-2 (top panel), matriptase (second panel from top), matriptase-HAI-1 complex formation (third panel from top, non-boiled/non- reduced) and prostasin (second panel from bottom). Tubulin was used as a loading control (bottom panel). Nonspecific bands are marked with n.s. C, conditioned medium from the cell samples from B was also analyzed using antibodies recognizing matriptase (top panel) and the matriptase-HAI-1 complex (lower panel). The specific proteins are indicated on the right, and the molecular markers are indicated on the left (kDa). The data are representative of five similar experiments.

Article Snippet: Before applying the goat anti-mouse HAI-2 antibody to the PVDF membrane for overnight probing, the antibody was pre-absorbed with 10 g of protein from HAI-2 knock-out lysate for 30 min at 4 °C in 1% nonfat dry milk/PBS-T. Antibodies used for Caco-2 cell lysates were sheep anti-human matriptase (AF3946, R&D Systems), mouse anti-human matriptase monoclonal antibody M69 (39), goat anti-human HAI-1 (AF1048, R&D Systems), polyclonal rabbit anti-human SPINT2 (HPA011101, Sigma) and mouse anti-human prostasin (catalogue no. 612173, BD Transduction Laboratories).

Techniques: Activation Assay, Binding Assay, Western Blot, SDS Page, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

doi: 10.1074/jbc.m114.574400

Figure Lengend Snippet: FIGURE 6. Reduction of HAI-2 levels in intestinal epithelial cell monolayers reduces the expression and alters the subcellular localization of matrip- tase. Caco-2 cells were transfected with matriptase siRNA (siST14, A–D), control siRNA (siCTRL, E–H) or siRNA targeting HAI-2 (siSPINT2, I–L), and the cells were grown on transwell filters. Three days post-transfection, the siRNA transfected samples were fixed and co-stained with antibodies against occludin (A, E, I) and matriptase (B, F, J). Phalloidin was used as an apical marker to verify the polarization of the monolayer (C, G, K). All three stainings were merged in D, H, and L. Images were taken in the XY plane, showing a single section through the monolayer, and in the XZ plane, showing a cross-section of the monolayer using confocal imaging. The position of the XY section is indicated on the XZ section (black arrows) on the right. Scale bar, 10 m. The data are representative of four similar experiments.

Article Snippet: Before applying the goat anti-mouse HAI-2 antibody to the PVDF membrane for overnight probing, the antibody was pre-absorbed with 10 g of protein from HAI-2 knock-out lysate for 30 min at 4 °C in 1% nonfat dry milk/PBS-T. Antibodies used for Caco-2 cell lysates were sheep anti-human matriptase (AF3946, R&D Systems), mouse anti-human matriptase monoclonal antibody M69 (39), goat anti-human HAI-1 (AF1048, R&D Systems), polyclonal rabbit anti-human SPINT2 (HPA011101, Sigma) and mouse anti-human prostasin (catalogue no. 612173, BD Transduction Laboratories).

Techniques: Expressing, Transfection, Control, Staining, Marker, Imaging

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

doi: 10.1074/jbc.m114.574400

Figure Lengend Snippet: FIGURE 7. Enhanced matriptase activation and shedding caused by reduction of HAI-2 levels is prostasin-dependent. A, HEK293 cells were co-trans- fected with pSRE-firefly luciferase and pRL-Renilla luciferase reporter plasmids in combination with expression vectors for PAR-2 (lanes 1–8), matriptase (lanes 1–4), prostasin (lanes 1–2 and 5–6), and the doxycycline inducible rtTA2S-M2 tet-transactivator together with the pBigHAI-2 expression vector containing a tet-responsive element (lanes 1–8). Expression of HAI-2 was induced with doxycycline in lanes 2, 4, 6, and 8. Data are shown as the mean S.D. of triplicate transfections of firefly luciferase units/Renilla luciferase units. *, p 0.05; **, p 0.005; ***, p 0.0005, Student’s t test, two-tailed. The data are representative of three similar experiments. B, HEK293T cells were co-transfected with matriptase (lanes 1–4), prostasin (lanes 1–4) and either HAI-2 (lanes 1 and 3) or HAI-2 containing an HA-tag (lanes 2 and 4). The cells were lysed 48 h after transfection and a fraction of the lysate (lanes 1 and 2) was analyzed by SDS-PAGE under reducingconditionsfollowedbyWesternblotwithHAI-2(toppanel),prostasin(middlepanel),andmatriptase(bottompanel)antibodiestoverifytheexpression of the various proteins. The remaining lysate was used for immunoprecipitation (IP, lanes 3 and 4) with HA-antibodies and the precipitated proteins were subjected to SDS-PAGE and Western blot as for the lysates. HAI-2HA was successfully precipitated (top panel, lane 4) and prostasin efficiently co-precipitated with HAI-2HA (middle panel, lane 4). The 95 kDa pro-form of matriptase was also co-precipitated with HAI-2 HA (bottom panel, lane 4). The specific proteins are indicated on the right, and the molecular markers are indicated on the left (kDa). The data are representative of two similar experiments. C, Caco-2 cells were transfected with control siRNA (siCTRL, lanes 1 and 4), siRNA targeting prostasin (siPRSS8, lanes 2 and 5) or HAI-2 (siSPINT2, lanes 3 and 6). Three days post- transfection the cells were lysed and a fraction of the lysate (lysate, lanes 1–3) was analyzed by SDS-PAGE and Western blot using antibodies against HAI-2 (top panel)andprostasin(bottompanel).Theremaininglysatewasusedforimmunoprecipitation(IP,lanes4–6)withHAI-2-antibodiesandtheprecipitatedproteins were subjected to SDS-PAGE and Western blot as for the lysates. Prostasin was efficiently co-precipitated with HAI-2. The specific proteins are indicated on the right, and the molecular markers are indicated on the left (kDa). The data are representative of two similar experiments. D and E, siRNAs for silencing down the gene of HAI-2 (siSPINT2, lanes 3 and 4), two prostasin-silencing siRNAs (siPrss8 a and b, lanes 5–8) and a combination hereof (lanes 9–12) where transfected into Caco-2 cells. Scrambled siRNA was used as control (siCTRL, lanes 1–2). All samples were loaded in duplicates. D, three days post-transfection, the cell lysates were analyzed by Western blot using antibodies against HAI-2 (top panel), prostasin (second panel from top), matriptase (third panel from top), and the matriptase-HAI-1 complex (second panel from bottom, non-boiled/non-reduced). Tubulin was used as a loading control (bottom panel). E, conditioned media from the cell samples from B was also analyzed using an antibody recognizing matriptase (top panel) and the matriptase-HAI-1 complex (bottom panel). The specific proteins are indicated on the right and the molecular markers are indicated on the left (kDa). The data are representative of four similar experiments. F–Q, Caco-2 cells were transfected with scrambled siRNA as control (siCTRL, F–H) or siRNA against HAI-2 (siSPINT2, I–K), prostasin (siPRSS8, L–N), or both (siPRSS8siSPINT2, O-Q). Three days post-transfection, the siRNA transfected samples were fixed and co-stained with antibodies against occludin (F, I, L, O) and matriptase (G, J, M, Q). The staining was merged in H, K, N, and P. Images were taken in the XY plane, showing a single section through the monolayer, and in the XZ plane showing a cross section of the monolayer using confocal imaging. The position of the XY section is indicated on the XZ section (black arrows) on the right. Scale bar, 10 m. The data are representative of two similar experiments.

Article Snippet: Before applying the goat anti-mouse HAI-2 antibody to the PVDF membrane for overnight probing, the antibody was pre-absorbed with 10 g of protein from HAI-2 knock-out lysate for 30 min at 4 °C in 1% nonfat dry milk/PBS-T. Antibodies used for Caco-2 cell lysates were sheep anti-human matriptase (AF3946, R&D Systems), mouse anti-human matriptase monoclonal antibody M69 (39), goat anti-human HAI-1 (AF1048, R&D Systems), polyclonal rabbit anti-human SPINT2 (HPA011101, Sigma) and mouse anti-human prostasin (catalogue no. 612173, BD Transduction Laboratories).

Techniques: Activation Assay, Luciferase, Expressing, Plasmid Preparation, Transfection, Two Tailed Test, SDS Page, Immunoprecipitation, Western Blot, Control, Staining, Imaging