PMC

Prostasin has important roles in chemoresistance and cell death in cell culture model. (a) Prostasin decreased in paclitaxel-resistance cancer cell line. O432: ovarian cancer cell line Ovca432 (sensitive to paclitaxel); O432-RP: paclitaxel resistance cell line generated from Ovca432. The prostasin protein levels in O432 and O432-RP cells are shown in immunoblots with specific antibodies. (b) Prostasin siRNAs transfection reduced prostasin. O432-pro-D cells (transfected with prostasin siRNA) express lower prostasin, compared with control cells of O432 (transfected with reagent only) and O432-Cs (transfected with no-targeting siRNA). The prostasin protein levels are shown in immunoblots with specific antibodies. (c) Downregulation of prostasin in O432 cells resulted in increase of chemoresistant activity. Cells were treated with paclitaxel at different concentrations for 24 h (starting 48 h after siRNAs transfection) and cultured with normal medium for an additional 7 to 10 days before cell survival was assayed. Relative cell survival rates of each cell line are shown. (d) Overexpression of prostasin greatly induces cell death in ovarian cancer cells. The cell survival rates are shown after forced overexpression of prostasin in several cell lines from day-0 to day-8, respectively. (e) Prostasin cDNA transfection resulted in overexpression of prostasin in chemoresistant O432-RP cells. O432-RP-pro-O cells (transfected with prostasin cDNA) express higher prostasin compared with control cells O432-RP and O432-RP-C (transfected with control vector). The prostasin protein levels are shown in immunoblots with specific antibodies. (f) Forced overexpression of prostasin represses growth of chemoresistant cells. Relative cell growth rates are shown for O432-RP-pro-O and control cells O432-RP and O432-RP-C. (g) Forced overexpression of prostasin in O432-RP cells re-sensitizes chemoresistant cells. Cells were plated at about 10–20% confluence and treated with paclitaxel at different concentrations for 24 h, cultured with normal medium for additional 7 to 10 days, then assayed for cell survival. Relative survival rates of cell lines are shown

A. The prostasin containing species present in Caco-2 cell lysates were separated and purified by sequential immunoaffinity chromatography using HAI-1 mAb M19-Sepharose, HAI-2 mAb DC-16 Sepharose, and then prostasin mAb YL11-Sepharose. The purified prostasin-HAI-1 complexes (lane 1), prostasin-HAI-2 complexes (lane 2), and prostasin monomers (lane 3) were analyzed for prostasin species by immunoblot using the prostasin-specific mAb YL11. B. Purified prostasin monomers (lane 1) were incubated with purified HAI-1 (lane 2) at 37°C for 5 min to allow the formation of prostasin-HAI-1 complexes (lane 3). The samples were then analyzed by western blot for prostasin-containing species. C. Purified prostasin monomers were incubated with a fluorogenic prostasin substrate and the kinetics of AMC release was recorded. Prostasin proteolytic activity assays were conducted at least four times. Representative data are shown. RFU stands for relative fluorescent unit.

Human foreskin lysates were prepared from 4 different donors in two batches with specimen 1–3 together and specimen 4 alone. Equal amount of protein from the four tissue lysates were analyzed by immunoblot for prostasin, matriptase, and HAI-1, as indicated (A and B). HAI-2 species were also analyzed in specimens 1–3 (C). The prostasin species include prostasin zymogen, active prostasin, and prostasin complex with HAI-1, as indicated. Matriptase species include matriptase zymogen and matriptase complex with HAI-1, as indicated. HAI-1 species include HAI-1 monomer, HAI-1 complexes with prostasin or matriptase, as indicated. HAI-2 species include the one with heavy N-glycan (C, right panel) and the one with light N-glycan (C, left panel).

Severe skin phenotype in transgenic mice with epidermal overexpression of wild type prostasin, zymogen-locked prostasin, and catalytically inactive prostasin. A–C, representative examples of the outward appearance of 12–15-day-old transgenic founders expressing wild type prostasin (A), zymogen-locked prostasin (B), and catalytically inactive prostasin (C) under the control of a keratin-5 promoter. A littermate without phenotype is shown on the left in A, and non-transgenic littermates are shown on the left for comparison in B and C. Note severe growth retardation, abnormal pelage hair, and skin ulcerations in transgenic founders expressing all three forms of prostasin. Shown are hematoxylin and eosin staining (D, G, J, and M), prostasin immunohistochemistry (E, H, K, and N), and Ki-67 immunohistochemistry (F, I, L, and O) of representative sections of dorsal skin from non-transgenic founders (D–F) and from founders expressing wild type (G–I), zymogen-locked (J–L), and catalytically inactive (M–O) prostasin under control of a keratin-5 promoter. The thickness of the epidermis is indicated by the vertical bar in D, G, J, and M, and the position of the basement membrane is indicated by the dotted line. Note the epidermal thickening and hyperkeratosis in wild type, zymogen-locked, and catalytically inactive prostasin transgenic founders, as compared with non-transgenic littermates. Insets in H, K, and N, high magnification images showing membrane-localized prostasin staining in keratinocytes. Examples of Ki-67-positive (proliferating) basal keratinocytes are shown with arrows in F, I, L, and O, and examples of proliferating suprabasal keratinocytes are shown with arrowheads in I, L, and O. Note the severe epidermal hyperproliferation in wild type prostasin, zymogen-locked prostasin, and catalytically inactive prostasin transgenic founders when compared with non-transgenic skin. Size bars, 50 μm (large panels) and 10 μm (insets).

Characterization of stably transfected M-1 cell lines. M-1 cells were stably transfected with pcDNA3.1 empty vector (control), wild-type prostasin (mPR-wt), catalytically inactive prostasin (mPR-S238A), or chimeric transmembrane (TM), non-GPI-anchored prostasin (mPR-Tpsg). Prostasin was assayed by immunoblotting. Samples were normalized to cell number for conditioned media and protein concentration for cell lysates. A: schematic diagrams of prostasin expression constructs. B: membrane association and GPI anchoring of prostasin variants. Cells were treated with PI-PLC or buffer alone, extracted in 60 mM n-octylglucoside-1% Triton X-100 in TBS on ice, resolved by SDS-PAGE, and assayed for prostasin by immunoblotting. mPR-Tpsg (arrowhead) is resistant to shedding by PI-PLC and migrates slightly faster than prostasin (arrow). C: apical (A) and basolateral (BL) secretion of prostasin variants from stably transfected, polyclonal M-1 cell lines cultured on 0.4-μm-pore filters. D: domain-selective cell surface biotinylation. M-1 cells were grown on 0.4-μm-pore Transwell filters until stable transepithelial resistance (R_te) developed. Cells were incubated with sulfo-NHS-biotin (0.5 mg/ml) or PBS control (data not shown) in apical or basolateral chamber. Biotinylated proteins were captured with streptavidin-agarose, resolved by 10% SDS-PAGE, and assayed for prostasin by immunoblotting. E: M-1 cells were transfected with pcDNA3.1-mPR-FLAG (diagram of construct at bottom of figure) or control vector. Conditioned medium was assayed for prostasin by immunoblot with anti-prostasin antibody (left), immunoprecipitation with anti-FLAG M2 antibody followed by immunoblot with prostasin antibody (middle), or immunoblot with M2 antibody after cell surface proteins were isolated by biotinylation and streptavidin-agarose affinity capture (right). F: M-1 cells were transfected with pcDNA3.1-mPR-Tpsg, pcDNA3.1-mPR-wt, pcDNA4-mPR-Tpsg-V5 (diagram of construct at bottom of figure), or control. Cell lysates and conditioned media were assayed by immunoblot using V5 antibody (top) or prostasin antibody (bottom).

Targeted HAI-2 deletion causes increased prostasin proteolysis with increased lifespan of active prostasin in Caco-2 cells. (A) Lysates were prepared from parental Caco-2 cells (Parental, lane 1) and two representative clones (KO1 and KO2, lanes 2 and 3), and equal amounts of protein were analyzed by western blot for prostasin expression using the mAb YL11. (B) through (F) The HAI-2 KO1 Caco-2 cells were engineered to express HAI-2 (C, D; HAI-2 Teton) or HAI-1 (E, F; HAI-1 Teton) or with empty vector as a control for doxycycline treatment (B; LVX-control) in a doxycycline-inducible manner. The Caco-2 variants were treated with increasing concentrations of doxycycline, as indicated, to induce expression of the HAIs. Lysates were prepared from these cells, and equal amounts of protein were analyzed by immunoblot for prostasin species using the prostasin mAb YL11 (B, D and F), for HAI-2 using the HAI-2 mAb CD16 (C) and for HAI-1 using the HAI-1 mAb M19 (E). The prostasin species, including the complexes with the HAIs (Prostasin Complexes with HAI-1 or HAI-2), enzymatically active prostasin (Active Prostasin) and the zymogen form (Zymogen Prostasin) are indicated. HAI-2 monomer and HAI-1 monomer are also indicated.

Mutant S/A prostasin is catalytically inactive. A: cartoon representation of a prostasin-aprotinin complex (Protein Database code 3GYM). His⁸⁵, Asp¹³⁴, and Ser²³⁸ in the catalytic triad of prostasin (green) are shown. Aprotinin is colored in red. B: amidolytic activity was assessed in culture media from human embryonic kidney (HEK)-293H cells transfected with green fluorescent protein (GFP), prostasin, or mutant S/A prostasin as described in experimental procedures. Amidolytic activity was significantly greater in media collected from HEK-293H cells transfected with prostasin than media collected from cells expressing GFP or mutant S/A prostasin. Statistically significant differences are indicated as *P < 0.05 and **P < 0.01 (n = 4) by ANOVA followed by Tukey's multiple-comparison test. Amidolytic activity in media collected from HEK-293H cells transfected with GFP was not significantly different than in media collected from cells expressing mutant S/A prostasin. C: prostasin secretion in media collected from HEK-293H cells transfected with GFP, wild-type prostasin, or mutant S/A prostasin. The presence of secreted wild-type and mutant prostasin in the media was assessed by IB. Numbers on the left of the image represent the mobility of Bio-Rad Precision Plus protein standards (in kDa). Data are representative of four independent experiments.

a Western blot analysis of TMPRSS13, matriptase, prostasin, and β-actin upon silencing with two non-overlapping RNA duplexes (siRNA 1 and siRNA 2) b Quantification of prostasin protein normalized to β-actin. c Invasion assays were performed in siRNA-treated HCC1937 cells 96 h post transfection with two non-overlapping synthetic RNA duplexes (siRNA-P1 and siRNA-P2) targeting prostasin and a %GC-matched RNA (in triplicates). Quantification of prostasin knockdown (KD) cells (red) and control cells (blue) invading through Matrigel (16 h) (upper panel) *p < 0.05, **p < 0.01 (one-way ANOVA, with Tukey’s post-hoc test for multiple comparisons). Western blot analysis to confirm prostasin silencing (96 h) (lower panel). Data are representative of three independent experiments. d Whole-cell protein lysates from HEK293T cells expressing empty expression vector (EV), expression vector with V5-tagged full-length human TMPRSS13 (TMPRSS13-V5) and EV, human full-length prostasin and EV, or TMPRSS13-V5 and prostasin were separated by SDS-PAGE under reducing conditions. Prostasin and β-actin were detected by western blotting. A lower molecular weight form of prostasin (cleaved prostasin?) is detected when co-transfected with TMPRSS13. e HEK293T cells expressing EV, prostasin/EV, or TMPRSS13-V5/prostasin, were immunoprecipitated with a mouse anti-V5 antibody and analyzed by western blotting (right panels) using the mouse anti-V5 antibody or a mouse anti-prostasin antibody. Whole-cell lysates were analyzed by western blotting to verity expression before precipitation (input, left panels). IgG H = IgG heavy chain detected by the secondary anti-mouse antibody. f HEK293T cells were transfected with full-length human wildtype prostasin (Pros-WT) and full-length human wildtype TMPRSS13 (T13-WT), T13-WT and zymogen locked prostasin (Pros-ZL), or Pros-WT and catalytically dead TMPRSS13 (T13-CD). Cells were treated with PI-PLC to release GPI-anchored prostasin 48 h post transfection. Released soluble prostasin was incubated with PN-1 (+) or buffer (−) for 1 h at 37 °C and samples were analyzed by SDS-PAGE under reducing conditions and western blotting using a mouse anti-prostasin antibody. The position of prostasin and prostasin-PN-1 complexes are indicated. A recombinant, soluble, truncated active form of purified prostasin protein (rProstasin) with or without PN-1 was included as positive control for complex formation. Lane 1: rProstasin; lane 2: rProstasin with addition of PN-1; lane 3: HEK293T cells expressing Pros-WT and T13-WT; lane 4: HEK293T cells expressing Pros-WT and T13-WT with addition of PN-1; lane 5: HEK293T cells expressing Pros-ZL and T13-WT; lane 6: HEK293T cells expressing Pros-ZL and T13-WT with addition of PN-1; lane 7: HEK293T cells expressing Pros-WT and T13-CD; lane 8: HEK293T cells expressing Pros-WT and T13-CD with addition of PN-1.

(a) Effect of prostasin on human CYP11B2 promoter activity in H295R cells. Cells were transfected with human CYP11B2 promoter-luciferase construct (pB2-1521) for 24 hours and then treated with vehicle or prostasin for another 24 hours. (b) Dose-dependent effect of prostasin on CYP11B2 mRNA expression in H295R cells. Cells were treated with increasing doses of prostasin for 24 hours. (c) Time course of CYP11B2 mRNA induction by prostasin in H295R cells. Cells were treated with 100 µg/mL of prostasin for 0, 3, 6, and 24 hours. (d) Dose-dependent effect of prostasin on aldosterone production in H295R cells. Cells were treated with increasing concentrations of prostasin for 48 hours. Values are means ± SD (n = 6). *P < .05, ^†P < .01, and ^#P < .001.

(A) Representative dot plot of PD-L1 (x-axis) and prostasin expression (y-axis) in Calu-3 cells treated with IFNγ (100 ng/ml for 24 h). Red: unstained cells. Green: secondary antibody (anti-rabbit-Cy2) and isotype IgG-APC. Magenta: prostasin antibody-labeled cells. Orange: PD-L1-APC antibody-labeled cells. Sky blue: prostasin and PD-L1 antibodies double-labeled cells. (B) Representative dot plot of prostasin expression in Calu-3 sublines. CC (red): Calu-3 control subline with scrambled gRNA, KO (green): Calu-3 knockout subline with prostasin-specific gRNA, V (magenta): Calu-3 subline with vector alone, P (orange): Calu-3 subline overexpressing the wildtype prostasin, M (sky blue): Calu-3 subline overexpressing an active-site mutant prostasin, G (blue): Calu-3 overexpressing an active prostasin without the GPI anchor. (C,D) Histogram of PD-L1 expression in Calu-3 sublines without IFNγ treatment (C) or with IFNγ treatment (D). (E) MFI of PD-L1 (filled boxes) and isotype antibody (open boxes) from duplicate settings were quantified and presented in bar graphs. Data are presented as mean ± SD. ANOVA P<0.05, * denotes P<0.05 as compared with the vector control (V).

(A–C) Immunohistochemical detection of prostasin at E8.5 in epithelial cells of surface ectoderm (examples with arrows in A and B) overlying the cranial neural tube region. Specificity of staining is shown by the absence of staining of Prss8^−/− surface ectoderm (arrow in C). Filled arrowhead shows non-specific staining of yolk sac. No expression was observed in the neuroepithelium (A and B, open arrowheads). (D–F) Immunohistochemical detection of prostasin in the chorionic ectoderm (examples with arrows) of mouse placenta at E8.5. Specificity of staining is shown by the absence of staining of Prss8^−/− chorionic ectoderm (F). Filled arrowheads in D and F shows non-specific staining of trophoblast giant cells. No expression was detected in the trophoblast stem cell-containing chorionic epithelium (open arrowhead in E). (G–I) Immunohistochemical detection of prostasin in the placental labyrinth (examples with arrows in G and H) of mouse placenta at E12.5. Specificity of staining is shown by the absence of staining of the Prss8^−/− labyrinth (I). No expression was detected in the trophoblast stem cell-containing chorionic epithelium (open arrowhead in H). Scale bars: A, C, D, F, G, and I, 100 µm; B, E, and H, 25 µm. (J) Enzymatic activity of wildtype (red), V170D (blue), S238A (grey), and zymogen (black) forms of prostasin. Prostasin variants were incubated with 50 µM pERTKR-AMC fluorogenic peptide at 37°C. V170D prostasin exhibited about 6% of the amidolytic activity of wildtype prostasin. No activity of catalytically inactive prostasin or prostasin zymogen was detected. (K) Western blot detection of SDS-stable complexes between prostasin and protein nexin-1 (PN-1). Wildtype zymogen (lanes 1 and 2), activated wildtype (lanes 3 and 4), V170D (frizzy) zymogen (lanes 5 and 6), activated V170D (lanes 7 and 8), S238A zymogen (lanes 9 and 10), and activated S238A (lanes 11 and 12) prostasin variants were incubated with (lanes 2, 4, 6, 8, 10, and 12) or without (lanes 1, 3, 5, 7, 9, and 11) 250 ng of recombinant human PN-1. Wildtype, but not V170D or S238A variants of prostasin formed SDS-stable complexes with PN-1. Positions of pro-prostasin, activated prostasin (migrating slightly faster than the zymogen due to removal of the 12 aa propeptide that is not detected after 4–12% SDS/PAGE with anti-prostasin antibody), and prostasin/PN-1 complexes are indicated. Positions of molecular weight markers (kDa) are shown on left.

(Green) Node 1: EGFR signaling, with Erbitux and lapatinib showing effects on prostasin regulation of PD-L1 expression. Node 2: IFNγ signaling via PKCα, with Gö 6976 and U0126 showing effects on the prostasin-mediated potentiation of PD-L1 induction by IFNγ. Node 3: Prostasin and PD-L1 co-localization in exosomes.

Prostasin induces matriptase zymogen activation and stimulates TEER development. A, prostasin depletion increases matriptase zymogen accumulation and decreases matriptase zymogen activation. Quantification of the relative amounts of zymogen and active matriptase in prostasin silenced (siP1 and siP2) Caco-2 monolayers compared with control siRNA (siCtl) cultures on day 6 determined by densitometric analysis of immunoblots from three independent transfection experiments. Data are shown as mean ± S.E. B, prostasin expression induces matriptase activation. Prostasin was co-expressed in HEK293T cells along with matriptase and HAI-1. Immunoblots of cell lysates reveal that prostasin cleaves the matriptase zymogen to liberate the 30-kDa active form. An asterisk indicates that the blot was overexposed to detect active matriptase. C, exogenous addition of recombinant prostasin induces matriptase activation. Confluent Caco-2 cultures grown on Transwell filters for 4 days were treated with 5 nm recombinant prostasin (rProstasin), which was added to either the basal (rProstasin-Basal) or apical (rProstasin-Apical) chamber of triplicate wells. Lysates were prepared at the indicated times and analyzed by immunoblot for matriptase zymogen, active matriptase, and GAPDH. D, TEER development after the addition of recombinant prostasin in C. The plot shows the average change in TEER over time (mean ± S.E. from triplicate wells), relative to the initial TEER (∼400 ohms·cm²). E, stimulation of TEER development is specific to prostasin. Caco-2 monolayers cultured on transwell filters for 4 days were treated with 5 nm of the indicated recombinant proteases. Plot shows average change in TEER over time (mean ± S.E.), relative to the initial TEER (initial TEER ∼ 580 ohms·cm²).

Caco-2 lysates (lanes 1) were subjected to immunodepletion to remove prostasin species (lanes 2), or HAI-1 species (lanes 3), or HAI-2 species (lanes 4), or both HAI-1 species and HAI-2 species together (lanes 5), as indicated. The lysates were analyzed by immunoblot for HAI-2 species (A.), prostasin species (B.), and HAI-1 species (C.). The identification of the prostasin species has been replicated at least twice and further verified by immunopurification in .

Sections of human skin, containing the epidermis, were immunostained with the prostasin mAb YL11 (A and B, Total Prostasin), the activated prostasin mAb YL89 (C and D, Activated Prostasin), and mouse IgG as a negative control (F, Mouse IgG). The tissues were counterstained with hematoxylin and eosin (E, H&E). Representative examples of the staining observed are presented. Scale bar: ~100 μm. n>20.

Three prostasin monoclonal antibodies were generated using purified prostasin-HAI-1 complex as the antigen. The proteins eluted from HAI-1 mAb M19-Sepharose (A.) or HAI-2 mAb DC16-Sepharose (B.) immunoaffinity columns were analyzed by immunoblot using the three prostasin mAbs, YL11, YL10, and YL89 under non-boiled, non-reducing conditions (lanes 1) or boiled, non-reducing conditions (lanes 2).

(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, χ²) 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.

Endogenous expression and posttranslational modifications of prostasin in M-1 kidney epithelial cells. A: immunoblot of native prostasin. Proteins were extracted from M-1 cells in 1% Triton X-100 or by detergent phase separation using 2% Triton X-114 (TX-114). Samples were normalized for cell number and separated by SDS-PAGE. B: immunoblot for prostasin in apical and basolateral conditioned media from M-1 cells grown to confluence [transepithelial resistance (R_te) > 1,000 Ω × cm²] on 0.4-μm-pore filters. C: glycosylphosphatidylinositol (GPI) anchoring of prostasin. M-1 cells were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or PBS. Prostasin was assayed in conditioned medium and detergent phase cell lysates by immunoblotting. D: deglycosylation of prostasin. M-1 cells were treated with PI-PLC, concentrated conditioned medium was incubated with protein N-glycosidase F (PNGase F), and proteins were separated by SDS-PAGE and immunoblotted to detect prostasin.

A Western blot appearance of wild-type and mutant prostasin in kidney tissue as well as a recombinant truncated murine prostasin (amino acids 30-289, predicted mass 28 kDa) with or without deglycosylation by PNGaseF. Note that deglycosylation leads to single clear bands, compatible with glycosylation of prostasin in vivo.
B Expression of prostasin as analyzed by Western blot from kidney lysates treated with PNGaseF for deglycosylation. Representative lanes with n=3 for each genotype.
C Densitometric analysis of the obtained bands from two Western blots with n=5-6 for each genotype.
D Urinary excretion of prostasin measured with an ELISA in 24 h urine samples.

Zymogen-locked matriptase induces epidermal prostasin processing. Protein extracts from skin (a), kidney (b), lung (c), and intestine (d) from newborn St14 ^zym/zym (lanes 1 and 2), St14 ^+/+ (lanes 3 and 4), and St14 ^–/– (lane 5) littermates were separated by capillary electrophoresis and probed with antibodies against matriptase (top panels), prostasin (middle panels), or β-actin (bottom panels). Lanes 6 and 7 in (a) are skin extracts from prostasin null (Prss8 ^–/–) and prostasin zymogen-locked (Prss8 ^zym/zym) mice, respectively. Zymogens of matriptase and prostasin are indicated with filled arrows, and the activated forms are indicated with open arrows. n.s. non-specific. Positions of molecular weight markers (kDa) are indicated on the left. e Representative example of quantification of activated prostasin (open arrow) and zymogen prostasin (filled arrows) in protein extracts from skin from a newborn St14 ^zym/zym mouse (top panel), St14 ^+/+ mouse (second panel from top), and a newborn St14 ^–/– mouse (second panel from bottom). Skin extracts from a newborn mouse expressing zymogen-locked (Prss8 ^zym/zym) endogenous prostasin is included as reference (bottom panel). f Ratio of activated prostasin to total prostasin in skin extracts from newborn St14 ^zym/zym (left bar, n = 7), St14 ^+/+ (middle bar, n = 7), and St14 ^–/– (right bar, n = 2) mice, quantified as in (e). Data are shown as mean ± SD. *P = 0.0011 was determined by one-way ANOVA, two-tailed. Additional file : Raw supporting data