Journal: PLoS ONE

Article Title: Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

doi: 10.1371/journal.pone.0062826

Figure Lengend Snippet: A. Human keratinocyte HaCaT cells were incubated either with PBS (C) as a control or a pH 6.0 buffer (Acid, A) to induce activation of matriptase. Cell lysates were analyzed by immunoblot for matriptase (MTP) or HAI-1 (HAI-1). B. HaCaT cells were incubated with a pH 6.0 buffer to induce matriptase activation. The cells and the conditioned buffer together (Combined), the cells alone (Cellular) and the conditioned buffer alone (Shed) were analyzed for matriptase activity using a matriptase synthetic fluorescent substrate, Boc-Gln-Ala-Arg-AMC. Data are representative of four independent experiments done under similar conditions. C. The conditioned buffer was subjected to immunoprecipitation with the activated matriptase mAb M69. The loading control (Loading), the unbound fraction (Unbound), and the eluent (Elution) were assayed for matriptase activity using the substrate, Boc-Gln-Ala-Arg-AMC. Data are representative of three independent experiments done under similar conditions. D. HaCaT cells were incubated with DPBS as control or a pH 6.0 buffer to induce matriptase activation. The conditioned buffer was collected and subjected to gelatin zymography.

Article Snippet: The mouse monoclonal antibody (mAb) M24 and rat mAb 21-9 recognize both latent and activated forms of matriptase , , , ; the rabbit polyclonal antibody matriptase/ST14 (Bethyl, Montgomery, TX) recognizes an epitope present in the serine protease domain of matriptase and is, therefore, able to detect the matriptase/serpin complex after reducing and heating the samples.

Techniques: Incubation, Activation Assay, Western Blot, Activity Assay, Immunoprecipitation, Zymography

Journal: PLoS ONE

Article Title: Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

doi: 10.1371/journal.pone.0062826

Figure Lengend Snippet: a. Pro-HGF was incubated with increasing amounts of active matriptase, as indicated, either with or without HaCaT cells at 37°C for 30 min. Samples were analyzed by immunoblot for HGF cleavage using an antibody directed against the beta subunit of HGF. b. Primary human keratinocytes were incubated with DPBS (No Activation) or a pH 6.0 buffer (Activation) to induce activation of matriptase, after which the buffer on the cells was adjusted to pH 7.5. Plasminogen (50 nM) was then added to the cells and the generation of plasmin was monitored by the cleavage of a plasmin synthetic fluorescent substrate, Boc-Val-Leu-Lys-AMC. Data are representative of three independent experiments done under similar conditions. c. Primary human keratinocytes were incubated with a pH 6.0 buffer to induce matriptase activation, followed by buffering to pH 7.5. Plasminogen (50 nM) was then added to the cells in the presence of the shed fractions (combined) or in the absence of the shed fractions (Cellular) or the shed fraction alone (Shed). Generation of plasmin was monitored by the cleavage of Boc-Val-Leu-Lys-AMC. Data are representative of four independent experiments done under similar conditions.

Article Snippet: The mouse monoclonal antibody (mAb) M24 and rat mAb 21-9 recognize both latent and activated forms of matriptase , , , ; the rabbit polyclonal antibody matriptase/ST14 (Bethyl, Montgomery, TX) recognizes an epitope present in the serine protease domain of matriptase and is, therefore, able to detect the matriptase/serpin complex after reducing and heating the samples.

Techniques: Incubation, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

doi: 10.1371/journal.pone.0062826

Figure Lengend Snippet: a. Human keratinocytes (HaCaT), mammary (184 A1N4) and prostate (RWPE1) epithelial cells were incubated with a pH 6.0 buffer to induce matriptase activation. The conditioned buffers (S) and the total cell lysates (T) were analyzed by immunoblot for total matriptase. Arrow indicates the 110-kDa matriptase complex. b. HaCaT cells were incubated with a pH 6.0 buffer for the indicated times (minutes). The cell lysates and the conditioned buffers were subjected to immunoblot analyses for total matriptase. c. Left panel: Purified 110-kDa matriptase complex was analyzed by SDS-PAGE under non-reducing and boiled conditions (NR, B). The protein bands were visualized by staining with ProtoBlue Safe. The protein bands a and b , as indicated, were subjected to protein identification by MS/MS (see for sequence data). Middle and right panels: HaCaT cells, pretreated with human serum, were induced to activate matriptase by pH 6.0 treatment. Cell lysates and the concentrated conditioned buffers were subjected to immunoblot analyses by immunoblot for total matriptase (Total MTP) and AT (AT).

Article Snippet: The mouse monoclonal antibody (mAb) M24 and rat mAb 21-9 recognize both latent and activated forms of matriptase , , , ; the rabbit polyclonal antibody matriptase/ST14 (Bethyl, Montgomery, TX) recognizes an epitope present in the serine protease domain of matriptase and is, therefore, able to detect the matriptase/serpin complex after reducing and heating the samples.

Techniques: Incubation, Activation Assay, Western Blot, Purification, SDS Page, Staining, Tandem Mass Spectroscopy, Sequencing

Journal: PLoS ONE

Article Title: Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

doi: 10.1371/journal.pone.0062826

Figure Lengend Snippet: A . HaCaT cells were incubated with the three AT preparations, as indicated, at 37°C for 5 min. The cellular retention of AT were analyzed by immunoblot analyses using an AT antibody. B . The AT-pretreated cells were then induced to activate matriptase by a pH 6.0 buffer. The cell lysates and the conditioned buffers were analyzed for matriptase species and AT species, as indicated. C . Matriptase activity in the conditioned buffer was also assessed by the cleavage of Boc-Gln-Ala-Arg-AMC. Data was done in triplicate and is a representative example of three independent experiments.

Article Snippet: The mouse monoclonal antibody (mAb) M24 and rat mAb 21-9 recognize both latent and activated forms of matriptase , , , ; the rabbit polyclonal antibody matriptase/ST14 (Bethyl, Montgomery, TX) recognizes an epitope present in the serine protease domain of matriptase and is, therefore, able to detect the matriptase/serpin complex after reducing and heating the samples.

Techniques: Incubation, Western Blot, Activity Assay

Journal: PLoS ONE

Article Title: Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

doi: 10.1371/journal.pone.0062826

Figure Lengend Snippet: HaCaT cells were incubated with increasing active matriptase, as indicated, at 37°C for 30 min. The conditioned media were collected and subjected to dot blot analysis with two different loaded volumes (1× and 2×) for syndecan-1. Data are representative of three independent experiments done under similar conditions.

Article Snippet: The mouse monoclonal antibody (mAb) M24 and rat mAb 21-9 recognize both latent and activated forms of matriptase , , , ; the rabbit polyclonal antibody matriptase/ST14 (Bethyl, Montgomery, TX) recognizes an epitope present in the serine protease domain of matriptase and is, therefore, able to detect the matriptase/serpin complex after reducing and heating the samples.

Techniques: Incubation, Dot Blot

Journal: PLoS ONE

Article Title: Antithrombin Regulates Matriptase Activity Involved in Plasmin Generation, Syndecan Shedding, and HGF Activation in Keratinocytes

doi: 10.1371/journal.pone.0062826

Figure Lengend Snippet: The subcellular distribution of matriptase (MTP), prostasin, and HAI-1 and the events following matriptase activation in the simple/polarized and the stratified epithelium are presented schematically. In simple/polarized epithelium, matriptase is targeted to the basolateral surface and prostasin is targeted to the apical surface with HAI-1 targeted to the both cell surface subdomains. The polarized expression of matriptase and prostasin is, however, likely not present in stratified epithelium, and as a result, matriptase gains an access to prostasin. When high level matriptase activation is induced, active matriptase is rapidly inhibited by nearby HAI-1 and the short-lived active matriptase can act on prostasin only in keratinocytes and not in polarized epithelial cells, due to the differential subcellular distributions of matriptase and prostasin between the two epithelia. Active prostasin is also rapidly inhibited by HAI-1 by forming a complex. A proportion of the active matriptase is shed from cell surface, an event that is evident in the stratified epithelial cells, and not in polarized epithelial cells. The shedding of active matriptase, the inhibition of active matriptase by AT bound to the cell via surface heparan sulfate proteoglycans, such as syndecans, is more obvious and important in stratified epithelial cells than in polarized epithelial cells. The active matriptase that escapes inactivation by HAI-1 or AT, can then act on its substrates, such as PAR-2, pro-uPA, pro-HGF, and syndecans.

Article Snippet: The mouse monoclonal antibody (mAb) M24 and rat mAb 21-9 recognize both latent and activated forms of matriptase , , , ; the rabbit polyclonal antibody matriptase/ST14 (Bethyl, Montgomery, TX) recognizes an epitope present in the serine protease domain of matriptase and is, therefore, able to detect the matriptase/serpin complex after reducing and heating the samples.

Techniques: Activation Assay, Expressing, Inhibition

Journal: PLoS ONE

Article Title: HATL5: A Cell Surface Serine Protease Differentially Expressed in Epithelial Cancers

doi: 10.1371/journal.pone.0087675

Figure Lengend Snippet: ( A ) Conditioned media samples from Pichia pastoris clones transfected with either the expression vector without protease insert (vector), with HATL5 serine protease domain cDNA (HATL5 #1 and #2) or matriptase serine protease domain cDNA were analyzed by western blotting using an anti-HATL5 antibody (left panel) or an anti-matriptase antibody (right panel). The positions of HATL5 (open arrow head) and matriptase serine protease domain (black arrow head) are indicated. ( B ) Samples from the conditioned media described in (A) were incubated at 37°C for 60 min with the synthetic chromogenic peptide Suc-Ala-Ala-Pro-Arg-pNA (100 µm) and the absorbance at 405 nm was recorded ( C ) 5 nM purified active recombinant HATL5 (white bars) or matriptase (black bars) serine protease domain was incubated at 37°C for 60 min with the synthetic chromogenic peptide MeOSuc-Glu-Val-Lys-Met-pNA (100 µm) in the absence or presence (inhibitor and substrate added concomitantly) of HAI-1 (60 nM), HAI-2 (40 nM), aprotinin (2 µm), leupeptin (20 µm), benzamidine (2 mM), or serpinA1 (60 nM). Enzyme activities for each enzyme are depicted relative to activity when no inhibitor was added.

Article Snippet: The expression of recombinant proteases in the conditioned media from individual yeast clones was analyzed by SDS-PAGE and western blotting using a rabbit anti-TMPRSS11b (Abcam, Cambridge, MA) or a rabbit anti-matriptase antibody (Bethyl Laboratories, Montgomery, TX).

Techniques: Clone Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Incubation, Purification, Recombinant, Activity Assay