human matriptase serine protease domain  (R&D Systems)

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 1. Expression analysis of matriptase, HAI-1, HAI-2, and prostasin in B cancer cells by reverse- transcription/qPCR (a,b), western blotting (c–e), and flow cytometry (f). (a) Bar graph of relative mRNA expression levels of matriptase (Mat), HAI-1, HAI-2, prostasin (Pro) in Daudi (n = 4), Namalwa (n = 5), Ramos (n = 3), Raji (n = 3), JeKo-1 (n = 3), and RS4;11 cells (n = 2) using GAPDH as the reference. The prostasin bars do not appear in the bar graph, as the actual qPCR readouts were registered as “N/A” by the instrument. (b) Bar graph of mRNA quantity ratio of HAI-2 to matriptase after normalization with the GAPDH level in each cell line in (a). (c) Western blotting images of matriptase (Ab: A300-221A), HAI-2, and GAPDH. Twenty micrograms of total protein from the cell lysate of each individual culture (including 2 repeats) were analyzed. Daudi, lanes 1–3; Namalwa, lanes 4–6; Ramos, lanes 7–9. Top panel, matriptase (Mat); middle panel, HAI-2; bottom panel, GAPDH. (d) Densitometry bar graph of relative protein quantities of matriptase and HAI-2 using GAPDH as the reference. (e) The quantitative ratio of HAI-2 to matriptase in each cell line. (f) Flow cytometry histogram of matriptase expression evaluation in Ramos cells. The Ramos cells (4 × 105) were labeled with the matriptase antibody as described in the Materials and Methods section. The matriptase-positive cells are shown in the PE-A subset (blue peak). Cells without the matriptase antibody labeling (red peak) were not detected in the PE-A subset and were used as the gating control.

Article Snippet: A purified recombinant human matriptase serine protease domain (r-Mat SPD, R&D Systems), when added (0.2 nM) in the Ramos cell culture, also reduced the quantity of the endogenous matriptase (Figure 6i, middle panel, lane 3) and the gelatinase activity of the endogenously expressed matriptase (Figure 6i, top panel, lane 3), suggesting that the soluble active matriptase can further activate more cellular matriptase zymogen.

Techniques: Expressing, Reverse Transcription, Western Blot, Cytometry, Flow Cytometry, Labeling, Antibody Labeling, Control

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 2. Prostasin exosomes reduce matriptase quantity in B cancer cells. (a) Western blot images of matriptase (Ab: A300-221A) in samples from the cell lysate (top panel) and the conditioned media (bottom panel) after incubation with prostasin exosomes (Pro) or exosomes without prostasin (KO). The Daudi cells (2 × 105 cells each) were incubated with the exosomes in 50 µL of OPTI-MEM I/2%FBS (lanes 1–4) or RPMI medium (lanes 5–8) overnight. One-half of each cell lysate or 40 µL of each media supernatant were analyzed. (b) Western blot images of GAPDH from (a). (c) Densitometry of relative intensities of matriptase in the cell lysate or media (d). Data presented are the average intensity of lanes 1, 3, 5, 7 versus that of lanes 2, 4, 6, 8 after normalization with GAPDH in (b). (e) Western blot images of matriptase (top panel; Ab: sc-365482) in the Daudi, Namalwa, and Ramos cells treated with exosomes isolated from the HEK293T cells. Cells (2.5 × 105) were co-cultured with prostasin exosomes (Pexo, lanes 3, 6, 9) or vector exosomes (Vexo, lanes 2, 5, 8) in 100 µL of OPTI-MEM I/2%FBS. Cells without exosomes (None, lanes 1, 4, 7) were cultured in the same conditions. Bottom, GAPDH western blot image. (f) Bar graph of (e) expressed as the relative intensities of matriptase

Article Snippet: A purified recombinant human matriptase serine protease domain (r-Mat SPD, R&D Systems), when added (0.2 nM) in the Ramos cell culture, also reduced the quantity of the endogenous matriptase (Figure 6i, middle panel, lane 3) and the gelatinase activity of the endogenously expressed matriptase (Figure 6i, top panel, lane 3), suggesting that the soluble active matriptase can further activate more cellular matriptase zymogen.

Techniques: Western Blot, Incubation, Isolation, Cell Culture, Plasmid Preparation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 3. B cell matriptase quantity reduction by wild-type prostasin. (a) Western blot images of matriptase (Ab: sc-365482) and GAPDH in the Daudi (top two panels), Ramos (middle two panels), and Namalwa (bottom two panels) cells treated with exosomes isolated from the Calu-3 cells and sublines with over-expressed prostasin or variants. Calu-3, parent cells; KO, subline with prostasin

Article Snippet: A purified recombinant human matriptase serine protease domain (r-Mat SPD, R&D Systems), when added (0.2 nM) in the Ramos cell culture, also reduced the quantity of the endogenous matriptase (Figure 6i, middle panel, lane 3) and the gelatinase activity of the endogenously expressed matriptase (Figure 6i, top panel, lane 3), suggesting that the soluble active matriptase can further activate more cellular matriptase zymogen.

Techniques: Western Blot, Isolation

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 5. Ectopic expression of prostasin in B cancer cells. (a) Western blot analysis of transient expression of prostasin (P) or vector alone (V) in the Daudi, Namalwa, and Ramos cells. The lysate from 2 × 105 cells of each type was analyzed. Top panel, matriptase (Ab: sc-365482); middle panel, prostasin; bottom panel, GAPDH. (b) Flow cytometry analysis of Namalwa sublines with tetracycline-induced prostasin expression or vector alone. Red peak (vector-alone cells) and sky-blue peak (prostasin-expressing cells) are samples without the prostasin antibody incubation. Orange peak (vector-alone cells) and green peak (prostasin-expressing cells) are samples incubated with the prostasin antibody. All samples were incubated with a secondary antibody conjugated with the fluorophore Cy3, and 10,000 cells of each sample were analyzed in a CytoFLEX S flow cytometer. The data were analyzed with FlowJo™software v10.8.1 and are presented in the histogram. (c) Western blot analysis of NamalwaTR sublines. One hundred thousand cells of each sample were analyzed. Lanes 1 and 4 or V, samples of the vector control subline; lanes 2 and 5 or P, samples of the subline with the wild-type prostasin; lanes 3 and 6 or M, samples of the subline with a serine active-site mutant prostasin. Left panel, cells were grown in OPTI-MEM I/2%FBS with 1 µg/mL tetracycline (with tet); right panel, cells were grown without tetracycline (no tet) for 8 days. Top two panels, matriptase antibody (sc-365482); bottom two panels, prostasin antibody. (d) Western blot analysis of tet-conditioned media from (c). Two hundred milliliters of the conditioned media were precipitated with trichloroacetic acid (TCA) (final 16.7%) at 4 ◦C overnight. The pellet was collected via centrifu- gation and analyzed. The membrane was blotted with the AF3946 human matriptase/ST14 catalytic domain antibody.

Article Snippet: A purified recombinant human matriptase serine protease domain (r-Mat SPD, R&D Systems), when added (0.2 nM) in the Ramos cell culture, also reduced the quantity of the endogenous matriptase (Figure 6i, middle panel, lane 3) and the gelatinase activity of the endogenously expressed matriptase (Figure 6i, top panel, lane 3), suggesting that the soluble active matriptase can further activate more cellular matriptase zymogen.

Techniques: Expressing, Western Blot, Plasmid Preparation, Flow Cytometry, Incubation, Cytometry, Software, Control, Mutagenesis, Membrane

Journal: Cancers

Article Title: Exosome-Mediated Activation of the Prostasin-Matriptase Serine Protease Cascade in B Lymphoma Cells.

doi: 10.3390/cancers15153848

Figure Lengend Snippet: Figure 6. Impact of prostasin–matriptase cascade activation on B cancer cells. (a) Bar graph of cell count for two consecutive days of B cells treated with exosomes. Namalwa, n = 7; Ramos, n = 6; Raji, n = 5; Jeko-1, n = 6. * denotes p < 0.05. (b) Growth curves of NamalwaTR-Vec and NamalwaTR-Pro cells under tetracycline induction. Left graph, cells were set at 2.5 × 105/mL on day 0 and cultured in the growth medium containing 10%FBS for 4 days. Right graph, on day 4 (reset, indicated by the arrow), the cells were diluted in OPTI-MEM I/2%FBS to 5 × 105/mL and cultured for another 5 days. Tetracycline at 1 µg/mL was added into the culture on day 0 and maintained through culturing. n = 4 for each cell line, and * denotes p < 0.05. (c) Trypsin-like serine protease activity in the conditioned media of NamalwaTR-Vec and NamalwaTR-Pro cells (n = 4). Data were analyzed in Excel with student’s t test. * denotes p < 0.05 between the two sample groups. (d) Bar graph of annexin-V-positive cells analyzed by flow cytometry. Cells under tetracycline induction were cultured for various times (week 1, n = 3; week 2, n = 4; week 3, n = 3) and subjected to direct labeling of annexin V conjugated with fluorophore allophycocyanin (APC). Ten thousand cells for each sample were analyzed on the CytoFLEX S flow cytometer. Propidium iodide staining and FSC/SSC discrimination were used for gating the live singlets, which were further analyzed for annexin V staining. (e) Bar graph of migrated cells treated with exosomes for 24 h. * denotes p < 0.05. (f) Bar graph of migrated Namalwa sublines with the induction of prostasin expression for 2–4 days and reconditioned in RPMI medium for 1 day before seeding in Transwells for migration (n = 7). * denotes p < 0.05. (g) Bar graph of invaded cells treated with exosomes for 24 h. (h) Bar graph of invaded Namalwa sublines (n = 5) treated as in (f). * denotes p < 0.05. (i) Gelatin zymography and western blot analysis. Top panel, the Ramos cells (2 × 106) in 500 µL of RPMI/0.1%BSA were treated with vector exosomes (Vexo), prostasin exosomes (Pexo), or a purified recombinant human matriptase serine protease domain (r-Mat SPD) overnight. One-fifth of the cell lysate (lanes 1–3) or 20 µL of the conditioned medium (lanes 4–6) were analyzed. The Vexo or Pexo exosomes or the r-Mat SPD alone were incubated in RPMI/0.1%BSA and used as controls (lanes 7–9). The clear bands at ~70 kDa marked by a filled arrow are matriptase. These were recognized by matriptase antibodies (middle panel). Unidentified bands with gelatinase activity marked at * locations in lanes 4, 5, 7, 8 are inherited from the exosomes, as shown in the samples with the exosomes alone (lanes 7 and 8). The band marked by the white circle is unknown. Bands at ~28 kDa marked by an unfilled arrow are r-Mat SPD. Bottom panel is GAPDH, which is detected only in the cell lysate, not in media samples or the controls without cells. (j) Gelatin zymography of B cancer cells treated as described in (i). The matriptase gelatinase activity is decreased in the cell lysate (lanes 2, 6, 10, 14) but increased in the corresponding media samples (lanes 4, 8, 12, 16) upon Pexo treatment in comparison to that of the Vexo-treated samples (in lysate, lanes 1, 5, 9, 13; in media, lanes 3, 7, 11, 15), correspondingly.

Article Snippet: A purified recombinant human matriptase serine protease domain (r-Mat SPD, R&D Systems), when added (0.2 nM) in the Ramos cell culture, also reduced the quantity of the endogenous matriptase (Figure 6i, middle panel, lane 3) and the gelatinase activity of the endogenously expressed matriptase (Figure 6i, top panel, lane 3), suggesting that the soluble active matriptase can further activate more cellular matriptase zymogen.

Techniques: Activation Assay, Cell Counting, Cell Culture, Activity Assay, Cytometry, Labeling, Staining, Expressing, Migration, Zymography, Western Blot, Plasmid Preparation, Recombinant, Incubation, Comparison

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