sheep anti human matriptase pab  (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: