human st14 protease catalytic domain  (R&D Systems)

Journal: BMC Research Notes

Article Title: Matriptase cleaves the amyloid-beta peptide 1–42 at Arg-5, Lys-16, and Lys-28

doi: 10.1186/s13104-018-4040-z

Figure Lengend Snippet: Cleavage of Aβ1–42 by matriptase. a Synthetic human Aβ42 (1 µg, Bio-Techne) was mixed with a recombinant human matriptase protease domain (MatPD, “+” = 0.1 µg, “+++” = 0.3 µg, Bio-Techne) in a total volume of 20 µl with reaction buffer. Samples containing Aβ42 or MatPD alone were also prepared. The reactions were carried out at 37 °C for either overnight or 1.5 h, as indicated, before the samples were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue. b Duplicate samples as described for lanes 1 and 3 were sent for MALDI-TOF MS analysis, performed by the University of Florida Proteomics & Mass Spectrometry Core on a 4700 Proteomics Analyzer (Applied Biosystems). Peptide fragments generated by MatPD from Aβ42 were identified in the data graph from Sample 3 (Aβ42 + MatPD), as shown by the red boxes with the calculated (average) and observed masses listed in the table. Insets: left—separation of peaks “L17-K28” and “H6-K16” by MSight ; right—spectrum from Sample 1 (Aβ42 alone) set to the same intensity scale and in the same mass range. Other boxed peaks were also examined by MSight over the corresponding mass spectrum ranges between Samples 3 and 1 (not shown)

Article Snippet: A recombinant human matriptase protease domain (MatPD, Cat. No. 3946-SEB-010) and a synthetic human Aβ1–42 peptide (Cat. No. 1428/100U) were purchased from Bio-Techne (Minneapolis, MN).

Techniques: Recombinant, SDS Page, Staining, Mass Spectrometry, Generated

Journal: BMC Research Notes

Article Title: Matriptase cleaves the amyloid-beta peptide 1–42 at Arg-5, Lys-16, and Lys-28

doi: 10.1186/s13104-018-4040-z

Figure Lengend Snippet: Cleavage of APP in cultured cells by matriptase. a The APP695-EGFP (lanes 1, 3, 5, and 7) or the APP-R601A-EGFP mutant plasmid (R601A, lanes 2, 4, 6, and 8) (0.6 µg for each per transfection) was co-transfected in HEK293 cells with an empty plasmid (pLVX-Puro) (Vec, lanes 1 and 2), or the cDNA coding for human matriptase (Mat, lanes 3 and 4), protease-dead matriptase (MatM, lanes 5 and 6), or mouse TMPRSS6 (lanes 7 and 8) (0.4 µg for each per transfection). Twenty micrograms of total cell lysate from each sample were resolved on SDS-PAGE and western-blotted with an EGFP antibody or a β-tubulin antibody. The 55-kDa matriptase-specific APP-EGFP CTF is marked by the red asterisk, and the 38-kDa matriptase-specific APP-EGFP CTF is marked by the red arrowhead. b An empty plasmid (pLVX-Puro) (Vec, lane 1), or the cDNA coding for human matriptase (Mat, lane 2) or protease-dead matriptase (MatM, lane 3) (1.5 µg for each per transfection) was transfected in M17 human neuroblastoma cells. Twenty micrograms of total cell lysate from each sample were resolved on SDS-PAGE and western-blotted with an APP antibody, a matriptase antibody, or a GAPDH antibody. The data shown here were representative from three independently repeated experiments

Article Snippet: A recombinant human matriptase protease domain (MatPD, Cat. No. 3946-SEB-010) and a synthetic human Aβ1–42 peptide (Cat. No. 1428/100U) were purchased from Bio-Techne (Minneapolis, MN).

Techniques: Cell Culture, Mutagenesis, Plasmid Preparation, Transfection, SDS Page, Western Blot

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: To detect prostasin/HAI and prostasin/PN-1 inhibitory complexes, PI-PLC-released pro-prostasin variants were left untreated or first activated by incubation with 10 nM human recombinant matriptase serine protease domain (R&D Systems) for 20 minutes at 37°C.

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