mouse anti-human matriptase antibody  (Fisher Scientific)

Journal: ACS chemical biology

Article Title: Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein

doi: 10.1021/acschembio.7b00715

Figure Lengend Snippet: Matriptase biosensor design. (a) ddRFP-based biosensor schematic. Following proteolytic linker cleavage, the fluorescent “A” copy separates from the stabilizing “B” copy, resulting in loss of “A” copy fluorescence. (b) Example time course plot of biosensor mechanism; addition of matriptase cleaves the biosensor and reduces fluorescence over time (red), while absence of matriptase retains fluorescence over time (blue). (c) Matriptase cleavable linker designs and sequence information. Design name is derived from number of amino acids flanking the scissile bond. Scissile bond is highlighted in red (Arg) and blue (Val). Each sequence shown is derived from the natural pro-macrophage stimulating protein (Pro-MSP) sequence. N-term = N-terminus; K1–4 = Kringle domains 1–4; SPH = Serine Protease Homologue.

Article Snippet: A total of 5 × 10 5 cancer cells were resuspended in cold 1xPBS with 1 mg mL −1 bovine serum albumin (0.1% BPBS) solution, containing a 1:100 dilution of mouse anti-human matriptase antibody (Fisher Scientific).

Techniques: Fluorescence, Sequencing, Derivative Assay

Journal: ACS chemical biology

Article Title: Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein

doi: 10.1021/acschembio.7b00715

Figure Lengend Snippet: Biosensor kinetic measurements and characterization. (a) Time course trajectories of biosensors B3 to B10 with matriptase and (b) velocity graphs in the presence of varying concentrations of matriptase (Matr.). (c) Western blot bands correspond to three possible reaction products detected through the C-terminal hexahistidine-tag: uncleaved biosensor (top band, 59.4 kDa), **hydolytically cleaved biosensor (middle band, 40.3 kDa), and cleaved biosensor (bottom band, 32.5 kDa). Copy “A” product is not detected due to lack of a His-tag. Note, two gels were used for this experiment as indicated. (d) Velocity profile for B4, B8, and B9 in the presence of different serine protease family members, relative to matriptase activity. Experiments performed in triplicate and reports mean values with standard deviation. Data are normalized for background conditions of biosensor without matriptase.

Article Snippet: A total of 5 × 10 5 cancer cells were resuspended in cold 1xPBS with 1 mg mL −1 bovine serum albumin (0.1% BPBS) solution, containing a 1:100 dilution of mouse anti-human matriptase antibody (Fisher Scientific).

Techniques: Western Blot, Activity Assay, Standard Deviation

Journal: ACS chemical biology

Article Title: Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein

doi: 10.1021/acschembio.7b00715

Figure Lengend Snippet: Application of B4 for measuring protease activity and inhibition. (a) B4 measurement of matriptase activity expressed on human A549 lung (blue), PC3 prostate (red), and MDA-MB-231 breast (green) cancer cell lines, compared with media alone (magenta); *p < 0.01 vs control. (b) B4 measurement of matriptase inhibition by soluble KD1 inhibitor. (c) B4 measurement of matriptase inhibition by yeast-displayed wild-type KD1 (green), KD1-R260A (magenta), or noninduced yeast controls with (red) or without (blue) matriptase.

Article Snippet: A total of 5 × 10 5 cancer cells were resuspended in cold 1xPBS with 1 mg mL −1 bovine serum albumin (0.1% BPBS) solution, containing a 1:100 dilution of mouse anti-human matriptase antibody (Fisher Scientific).

Techniques: Activity Assay, Inhibition, 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 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