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Meridian Life Science pcr 2 1 topo vector
Pcr 2 1 Topo Vector, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcr 2 1 topo vector/product/Meridian Life Science
Average 92 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pcr 2 1 topo vector - by Bioz Stars, 2020-08
92/100 stars

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Polymerase Chain Reaction:

Article Title: Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity
Article Snippet: .. The reaction contained 4 μL of purified PCR product, 1 μL of salt solution, 1 μL of pCR®2.1-TOPO vector, and 1 μL of water (Bioline, USA). .. The reaction mixture was then transformed into TOP10F Escherichia coli and positive clones were selected on agar containing X-gal and identified by PCR and restriction enzyme digestion.

Plasmid Preparation:

Article Title: Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity
Article Snippet: .. The reaction contained 4 μL of purified PCR product, 1 μL of salt solution, 1 μL of pCR®2.1-TOPO vector, and 1 μL of water (Bioline, USA). .. The reaction mixture was then transformed into TOP10F Escherichia coli and positive clones were selected on agar containing X-gal and identified by PCR and restriction enzyme digestion.

Purification:

Article Title: Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity
Article Snippet: .. The reaction contained 4 μL of purified PCR product, 1 μL of salt solution, 1 μL of pCR®2.1-TOPO vector, and 1 μL of water (Bioline, USA). .. The reaction mixture was then transformed into TOP10F Escherichia coli and positive clones were selected on agar containing X-gal and identified by PCR and restriction enzyme digestion.

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    Meridian Life Science sheep anti uromodulin
    Effects of pharmacological ROMK inhibition on <t>uromodulin</t> processing. ( a ) Transepithelial resistance (R te ) of primary cultures obtained from microdissected TALs (mTAL) of wild-type mice after 12 hours treatment with 100 µM bumetanide (NKCC2 blocker) or 5 mM BaCl 2 (K + channels blocker). ( b ) Representative Western blot of secreted and cellular uromodulin (UMOD) in mTAL cells. Apical medium was collected 12 hours before and after treatment with bumetanide or BaCl 2 (as in panel a). ( c ) Transepithelial resistance (R te ) of mTAL cells 20 minutes after treatment with either 30 µM or 60 µM VU591 (ROMK inhibitor). ( d ) Representative Western blot of secreted and cellular uromodulin in mTAL cells. Apical medium was collected 12 hours before and after treatment with VU591. Full length blot images can be found in Supplementary Figure S2 .
    Sheep Anti Uromodulin, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sheep anti uromodulin/product/Meridian Life Science
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    Meridian Life Science rotavirus vp7
    Schematic of plant expression plasmids pPHAP1301-HRVVP7-CTB and pPHAP1301-HRVVP7. bar encodes a resistance marker for glufosinate selection. (A) HRVVP7 coding sequence. (Gly 4 Ser) 3 is the linker between HRVVP7 and CTB . (B) HRVVP7 coding sequence. HRVVP7, human <t>rotavirus</t> <t>VP7;</t> CTB, cholera toxin B subunit; Phas , β-phaseolin storage protein; 35s, Cauliflower mosaic virus; LB, left border; RB, right border; nos , nopaline synthase.
    Rotavirus Vp7, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effects of pharmacological ROMK inhibition on uromodulin processing. ( a ) Transepithelial resistance (R te ) of primary cultures obtained from microdissected TALs (mTAL) of wild-type mice after 12 hours treatment with 100 µM bumetanide (NKCC2 blocker) or 5 mM BaCl 2 (K + channels blocker). ( b ) Representative Western blot of secreted and cellular uromodulin (UMOD) in mTAL cells. Apical medium was collected 12 hours before and after treatment with bumetanide or BaCl 2 (as in panel a). ( c ) Transepithelial resistance (R te ) of mTAL cells 20 minutes after treatment with either 30 µM or 60 µM VU591 (ROMK inhibitor). ( d ) Representative Western blot of secreted and cellular uromodulin in mTAL cells. Apical medium was collected 12 hours before and after treatment with VU591. Full length blot images can be found in Supplementary Figure S2 .

    Journal: Scientific Reports

    Article Title: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK

    doi: 10.1038/s41598-019-55771-x

    Figure Lengend Snippet: Effects of pharmacological ROMK inhibition on uromodulin processing. ( a ) Transepithelial resistance (R te ) of primary cultures obtained from microdissected TALs (mTAL) of wild-type mice after 12 hours treatment with 100 µM bumetanide (NKCC2 blocker) or 5 mM BaCl 2 (K + channels blocker). ( b ) Representative Western blot of secreted and cellular uromodulin (UMOD) in mTAL cells. Apical medium was collected 12 hours before and after treatment with bumetanide or BaCl 2 (as in panel a). ( c ) Transepithelial resistance (R te ) of mTAL cells 20 minutes after treatment with either 30 µM or 60 µM VU591 (ROMK inhibitor). ( d ) Representative Western blot of secreted and cellular uromodulin in mTAL cells. Apical medium was collected 12 hours before and after treatment with VU591. Full length blot images can be found in Supplementary Figure S2 .

    Article Snippet: Antibodies The following primary antibodies were used: sheep anti-uromodulin (K90071C, Meridian Life Science Inc., Cincinnati, OH, USA; 1:500 for WB and 1:300 for IF), mouse anti-uromodulin (CL 1032 A; Cedarlane Laboratories; for ELISA), mouse anti-flotillin-1 (BD 610821, BD Biosciences, Franklin Lakes, NJ, 1:500 for WB), rabbit anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:500 for WB), mouse anti-β actin (A5441, Sigma-Aldrich; 1:10 000 for WB, rabbit anti-NKCC2 (AB3562P, Merck-Millipore, Burlington, MA, USA; 1:500 for WB and 1:100 for IF), rabbit anti-ROMK (Penton et al ., MS under revision; 1:1000 for WB and 1:5000 for IF).

    Techniques: Inhibition, Mouse Assay, Western Blot

    Kcnj1 −/− mice show reduced urinary uromodulin. ( a ) Representative Western blot of urinary uromodulin (UMOD) in 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. Urinary creatinine was used as a loading control. ( b ) Enzyme-linked immunosorbent assay (ELISA) of urinary uromodulin in 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. Uromodulin concentration was normalized on urinary creatinine. ( c ) Western blot for uromodulin (UMOD) in control and deglycosylated (PNGase F treatment) urine samples from Kcnj1 +/+ and Kcnj1 −/− mice. Samples were denatured by heating and treated according to the manufacturer’s instructions. Full length blot images can be found in Supplementary Figure S2 .

    Journal: Scientific Reports

    Article Title: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK

    doi: 10.1038/s41598-019-55771-x

    Figure Lengend Snippet: Kcnj1 −/− mice show reduced urinary uromodulin. ( a ) Representative Western blot of urinary uromodulin (UMOD) in 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. Urinary creatinine was used as a loading control. ( b ) Enzyme-linked immunosorbent assay (ELISA) of urinary uromodulin in 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. Uromodulin concentration was normalized on urinary creatinine. ( c ) Western blot for uromodulin (UMOD) in control and deglycosylated (PNGase F treatment) urine samples from Kcnj1 +/+ and Kcnj1 −/− mice. Samples were denatured by heating and treated according to the manufacturer’s instructions. Full length blot images can be found in Supplementary Figure S2 .

    Article Snippet: Antibodies The following primary antibodies were used: sheep anti-uromodulin (K90071C, Meridian Life Science Inc., Cincinnati, OH, USA; 1:500 for WB and 1:300 for IF), mouse anti-uromodulin (CL 1032 A; Cedarlane Laboratories; for ELISA), mouse anti-flotillin-1 (BD 610821, BD Biosciences, Franklin Lakes, NJ, 1:500 for WB), rabbit anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:500 for WB), mouse anti-β actin (A5441, Sigma-Aldrich; 1:10 000 for WB, rabbit anti-NKCC2 (AB3562P, Merck-Millipore, Burlington, MA, USA; 1:500 for WB and 1:100 for IF), rabbit anti-ROMK (Penton et al ., MS under revision; 1:1000 for WB and 1:5000 for IF).

    Techniques: Mouse Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

    Kcnj1 fl/fl Pax8 Cre /+ mice show reduced urinary uromodulin with accumulation in the kidney. ( a ) Representative Western blot analysis for uromodulin (UMOD) and ROMK in urine and whole kidney lysates of Kcnj1 fl/fl Pax8 +/+ and Kcnj1 fl/fl Pax8 Cre/+ mice after doxycycline induction. β-actin was used as a loading control. ( b ) Immunofluorescence staining for uromodulin of Kcnj1 fl/fl Pax8 +/+ and Kcnj1 fl/fl Pax8 Cre/+ kidney cryosections (scale bar: 50 µm). Full length blot images can be found in Supplementary Figure S2 .

    Journal: Scientific Reports

    Article Title: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK

    doi: 10.1038/s41598-019-55771-x

    Figure Lengend Snippet: Kcnj1 fl/fl Pax8 Cre /+ mice show reduced urinary uromodulin with accumulation in the kidney. ( a ) Representative Western blot analysis for uromodulin (UMOD) and ROMK in urine and whole kidney lysates of Kcnj1 fl/fl Pax8 +/+ and Kcnj1 fl/fl Pax8 Cre/+ mice after doxycycline induction. β-actin was used as a loading control. ( b ) Immunofluorescence staining for uromodulin of Kcnj1 fl/fl Pax8 +/+ and Kcnj1 fl/fl Pax8 Cre/+ kidney cryosections (scale bar: 50 µm). Full length blot images can be found in Supplementary Figure S2 .

    Article Snippet: Antibodies The following primary antibodies were used: sheep anti-uromodulin (K90071C, Meridian Life Science Inc., Cincinnati, OH, USA; 1:500 for WB and 1:300 for IF), mouse anti-uromodulin (CL 1032 A; Cedarlane Laboratories; for ELISA), mouse anti-flotillin-1 (BD 610821, BD Biosciences, Franklin Lakes, NJ, 1:500 for WB), rabbit anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:500 for WB), mouse anti-β actin (A5441, Sigma-Aldrich; 1:10 000 for WB, rabbit anti-NKCC2 (AB3562P, Merck-Millipore, Burlington, MA, USA; 1:500 for WB and 1:100 for IF), rabbit anti-ROMK (Penton et al ., MS under revision; 1:1000 for WB and 1:5000 for IF).

    Techniques: Mouse Assay, Western Blot, Immunofluorescence, Staining

    AdenoCre-mediated deletion of Kcnj1 in primary mouse TAL cells. ( a ) Representative Western blot analysis for NKCC2 and ROMK in primary cultured (mTAL) cells obtained from microdissected TAL segments of Kcnj1 fl/fl kidneys. Cells were transduced with either an empty adenoviral vector (Mock) or an individual expressing Cre-recombinase (Adeno-Cre). β-actin was used as a loading control. ( b ) Representative Western blot analysis of secreted and cellular uromodulin (UMOD) from Kcnj1 fl/fl mTAL cells treated with Mock or Adeno-Cre. ( c ) Transepithelial resistance (R te ) of Kcnj1 fl/fl mTAL cells 5 days after Mock or Adeno-Cre transduction. ( d ) Immunofluorescence for uromodulin in Kcnj1 fl/fl mTAL cells after Mock or Adeno-Cre transduction. Full length blot images can be found in Supplementary Figure S2 .

    Journal: Scientific Reports

    Article Title: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK

    doi: 10.1038/s41598-019-55771-x

    Figure Lengend Snippet: AdenoCre-mediated deletion of Kcnj1 in primary mouse TAL cells. ( a ) Representative Western blot analysis for NKCC2 and ROMK in primary cultured (mTAL) cells obtained from microdissected TAL segments of Kcnj1 fl/fl kidneys. Cells were transduced with either an empty adenoviral vector (Mock) or an individual expressing Cre-recombinase (Adeno-Cre). β-actin was used as a loading control. ( b ) Representative Western blot analysis of secreted and cellular uromodulin (UMOD) from Kcnj1 fl/fl mTAL cells treated with Mock or Adeno-Cre. ( c ) Transepithelial resistance (R te ) of Kcnj1 fl/fl mTAL cells 5 days after Mock or Adeno-Cre transduction. ( d ) Immunofluorescence for uromodulin in Kcnj1 fl/fl mTAL cells after Mock or Adeno-Cre transduction. Full length blot images can be found in Supplementary Figure S2 .

    Article Snippet: Antibodies The following primary antibodies were used: sheep anti-uromodulin (K90071C, Meridian Life Science Inc., Cincinnati, OH, USA; 1:500 for WB and 1:300 for IF), mouse anti-uromodulin (CL 1032 A; Cedarlane Laboratories; for ELISA), mouse anti-flotillin-1 (BD 610821, BD Biosciences, Franklin Lakes, NJ, 1:500 for WB), rabbit anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:500 for WB), mouse anti-β actin (A5441, Sigma-Aldrich; 1:10 000 for WB, rabbit anti-NKCC2 (AB3562P, Merck-Millipore, Burlington, MA, USA; 1:500 for WB and 1:100 for IF), rabbit anti-ROMK (Penton et al ., MS under revision; 1:1000 for WB and 1:5000 for IF).

    Techniques: Western Blot, Cell Culture, Transduction, Plasmid Preparation, Expressing, Immunofluorescence

    Histological characterization, transcript analysis and uromodulin processing in Kcnj1 −/− mice. ( a ) Haematoxylin and eosin (H E) staining of 12 weeks Kcnj1 +/+ and Kcnj1 −/− kidneys. Scale bars: low magnification - 2 mm, high magnification - 100 μm ( b ) Quantitative PCR analysis of tubular marker genes in Kcnj1 −/− kidneys. Glom (Glomerulus): Nphs2 (Podocin); PT (Proximal Tubule): Slc5a2 - Sodium/glucose cotransporter 2 (SGLT2), Aqp1 - Aquaporin 1; TAL (Thick Ascending Limb): Slc12a1 - Na/K/Cl cotransporter 2 (NKCC2), Umod - Uromodulin; DCT (Distal Convoluted Tubule): Slc12a3 - Na/Cl cotransporter (NCC), Pvalb - Parvalbumin; CNT/CD (Connecting Tubule/Collecting Duct): Aqp2 - Aquaporin 2, Scnn1b - Sodium channel epithelial 1 beta subunit (β-ENaC), Scnn1g - Sodium channel epithelial 1 gamma subunit (γ-ENaC); Cldn16 - Claudin 16 and Clcnkb (Cl channel Kb) are localized in both TAL and DCT. ( c ) Representative Western blot analysis on whole kidney lysates of 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. β-actin was used as a loading control. ( d ) Representative Western blot analysis on kidney membrane fractions of 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. Flotillin-1 was used as a loading control, whereas GAPDH was used to test cytosolic contamination. ( e ) Representative Western blot analysis on kidney cytosolic fractions of 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. GAPDH was used as a loading control, whereas Flotillin-1 was used to test membrane contamination. ( f ) Immunofluorescence analysis for UMOD (green) and NKCC2 (red) in Kcnj1 +/+ and Kcnj1 −/− serial kidney sections. Scale bars: low magnification - 50 μm, high magnification - 15 μm. Full length blot images can be found in Supplementary Figure S2 .

    Journal: Scientific Reports

    Article Title: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK

    doi: 10.1038/s41598-019-55771-x

    Figure Lengend Snippet: Histological characterization, transcript analysis and uromodulin processing in Kcnj1 −/− mice. ( a ) Haematoxylin and eosin (H E) staining of 12 weeks Kcnj1 +/+ and Kcnj1 −/− kidneys. Scale bars: low magnification - 2 mm, high magnification - 100 μm ( b ) Quantitative PCR analysis of tubular marker genes in Kcnj1 −/− kidneys. Glom (Glomerulus): Nphs2 (Podocin); PT (Proximal Tubule): Slc5a2 - Sodium/glucose cotransporter 2 (SGLT2), Aqp1 - Aquaporin 1; TAL (Thick Ascending Limb): Slc12a1 - Na/K/Cl cotransporter 2 (NKCC2), Umod - Uromodulin; DCT (Distal Convoluted Tubule): Slc12a3 - Na/Cl cotransporter (NCC), Pvalb - Parvalbumin; CNT/CD (Connecting Tubule/Collecting Duct): Aqp2 - Aquaporin 2, Scnn1b - Sodium channel epithelial 1 beta subunit (β-ENaC), Scnn1g - Sodium channel epithelial 1 gamma subunit (γ-ENaC); Cldn16 - Claudin 16 and Clcnkb (Cl channel Kb) are localized in both TAL and DCT. ( c ) Representative Western blot analysis on whole kidney lysates of 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. β-actin was used as a loading control. ( d ) Representative Western blot analysis on kidney membrane fractions of 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. Flotillin-1 was used as a loading control, whereas GAPDH was used to test cytosolic contamination. ( e ) Representative Western blot analysis on kidney cytosolic fractions of 12 weeks Kcnj1 +/+ and Kcnj1 −/− mice. GAPDH was used as a loading control, whereas Flotillin-1 was used to test membrane contamination. ( f ) Immunofluorescence analysis for UMOD (green) and NKCC2 (red) in Kcnj1 +/+ and Kcnj1 −/− serial kidney sections. Scale bars: low magnification - 50 μm, high magnification - 15 μm. Full length blot images can be found in Supplementary Figure S2 .

    Article Snippet: Antibodies The following primary antibodies were used: sheep anti-uromodulin (K90071C, Meridian Life Science Inc., Cincinnati, OH, USA; 1:500 for WB and 1:300 for IF), mouse anti-uromodulin (CL 1032 A; Cedarlane Laboratories; for ELISA), mouse anti-flotillin-1 (BD 610821, BD Biosciences, Franklin Lakes, NJ, 1:500 for WB), rabbit anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:500 for WB), mouse anti-β actin (A5441, Sigma-Aldrich; 1:10 000 for WB, rabbit anti-NKCC2 (AB3562P, Merck-Millipore, Burlington, MA, USA; 1:500 for WB and 1:100 for IF), rabbit anti-ROMK (Penton et al ., MS under revision; 1:1000 for WB and 1:5000 for IF).

    Techniques: Mouse Assay, Staining, Real-time Polymerase Chain Reaction, Marker, Western Blot, Immunofluorescence

    Urinary uromodulin levels in Bartter type 2 patients. ( a ) Amino acid composition of ROMK, with mutations found in Bartter type 2 patients (yellow, missense; red, nonsense). TM: Transmembrane domain. ( b ) Box and whiskers plot of urinary uromodulin (UMOD) levels of Bartter type 2 patients, control relatives and a healthy reference population (age ≤ 20, eGFR ≥ 80). Median, 25 th and 75 th percentiles are indicated on the respective plot. ( c ) Representative Western blot analysis of UMOD in urine samples from Bartter type 2 patients and control individuals. Samples were loaded according to urinary creatinine. ( d ) Western blot analysis of UMOD in control and deglycosylated (PNGase F treatment) urine samples from Bartter type 2 patients and control relatives. Samples were denatured by heating and treated according to the manufacturer’s instructions. Full length blot images can be found in Supplementary Figure S2 .

    Journal: Scientific Reports

    Article Title: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK

    doi: 10.1038/s41598-019-55771-x

    Figure Lengend Snippet: Urinary uromodulin levels in Bartter type 2 patients. ( a ) Amino acid composition of ROMK, with mutations found in Bartter type 2 patients (yellow, missense; red, nonsense). TM: Transmembrane domain. ( b ) Box and whiskers plot of urinary uromodulin (UMOD) levels of Bartter type 2 patients, control relatives and a healthy reference population (age ≤ 20, eGFR ≥ 80). Median, 25 th and 75 th percentiles are indicated on the respective plot. ( c ) Representative Western blot analysis of UMOD in urine samples from Bartter type 2 patients and control individuals. Samples were loaded according to urinary creatinine. ( d ) Western blot analysis of UMOD in control and deglycosylated (PNGase F treatment) urine samples from Bartter type 2 patients and control relatives. Samples were denatured by heating and treated according to the manufacturer’s instructions. Full length blot images can be found in Supplementary Figure S2 .

    Article Snippet: Antibodies The following primary antibodies were used: sheep anti-uromodulin (K90071C, Meridian Life Science Inc., Cincinnati, OH, USA; 1:500 for WB and 1:300 for IF), mouse anti-uromodulin (CL 1032 A; Cedarlane Laboratories; for ELISA), mouse anti-flotillin-1 (BD 610821, BD Biosciences, Franklin Lakes, NJ, 1:500 for WB), rabbit anti-GAPDH (2118, Cell Signaling Technology, Danvers, MA, USA; 1:500 for WB), mouse anti-β actin (A5441, Sigma-Aldrich; 1:10 000 for WB, rabbit anti-NKCC2 (AB3562P, Merck-Millipore, Burlington, MA, USA; 1:500 for WB and 1:100 for IF), rabbit anti-ROMK (Penton et al ., MS under revision; 1:1000 for WB and 1:5000 for IF).

    Techniques: Western Blot

    Hepsin is the protease mediating uromodulin polymerisation in MDCK cells. ( A ) Confocal immunofluorescence analysis showing uromodulin (green), hepsin or prostasin (red) and E-cadherin (blue) (basolateral membrane marker) in polarised MDCK cells, as indicated. Upper panels represent the reconstruction on the xz axis of merged xy scans, for which a representative image is shown in lower panels. Both serine proteases co-localise with uromodulin on the apical plasma membrane of polarised MDCK cells. z stacks = 0.3 µm. A: apical, BL: basolateral. Scale bars, 5 µm. ( B ) Transcript levels of HPN and PRSS8 , as assessed by Real-Time qPCR in MDCK cells transfected with shRNA vectors, as indicated. Expression values (normalised to glyceraldehyde-3-phosphate dehydrogenase, GAPDH) are shown as relative to cells transfected with control vector. Expression of the proteases is specifically reduced in silenced cells. Bars indicate average ± s.e.m. **p

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Hepsin is the protease mediating uromodulin polymerisation in MDCK cells. ( A ) Confocal immunofluorescence analysis showing uromodulin (green), hepsin or prostasin (red) and E-cadherin (blue) (basolateral membrane marker) in polarised MDCK cells, as indicated. Upper panels represent the reconstruction on the xz axis of merged xy scans, for which a representative image is shown in lower panels. Both serine proteases co-localise with uromodulin on the apical plasma membrane of polarised MDCK cells. z stacks = 0.3 µm. A: apical, BL: basolateral. Scale bars, 5 µm. ( B ) Transcript levels of HPN and PRSS8 , as assessed by Real-Time qPCR in MDCK cells transfected with shRNA vectors, as indicated. Expression values (normalised to glyceraldehyde-3-phosphate dehydrogenase, GAPDH) are shown as relative to cells transfected with control vector. Expression of the proteases is specifically reduced in silenced cells. Bars indicate average ± s.e.m. **p

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Immunofluorescence, Marker, Real-time Polymerase Chain Reaction, Transfection, shRNA, Expressing, Plasmid Preparation

    Expression of the serine protease inhibitor HAI-1 effectively reduces uromodulin cleavage at the urinary site. ( A ) Immunofluorescence analysis showing uromodulin on the surface of MDCK cells co-expressing the serine protease inhibitor HAI-1 (Hepatocyte growth factor Activator Inhibitor-1), as indicated. HAI-1 expression essentially abolishes uromodulin polymerisation on the cell surface. Scale bar, 50 µm. ( B ) Representative Western blot analysis showing uromodulin and HAI-1 expression in cell lysates of transfected MDCK cells, as indicated. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is shown as a loading control. ( C ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by MDCK cells co-expressing HAI-1, as indicated. Densitometric analysis (average ± s.d. of 3 independent experiments, Figure 2—source data 2 ) shows the ratio between the short and the long uromodulin isoforms in the absence or presence of HAI-1 co-expression, as indicated. The serine protease inhibitor HAI-1 strongly reduces the amount of the short uromodulin isoform released in the culturing medium by MDCK cells. *p

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Expression of the serine protease inhibitor HAI-1 effectively reduces uromodulin cleavage at the urinary site. ( A ) Immunofluorescence analysis showing uromodulin on the surface of MDCK cells co-expressing the serine protease inhibitor HAI-1 (Hepatocyte growth factor Activator Inhibitor-1), as indicated. HAI-1 expression essentially abolishes uromodulin polymerisation on the cell surface. Scale bar, 50 µm. ( B ) Representative Western blot analysis showing uromodulin and HAI-1 expression in cell lysates of transfected MDCK cells, as indicated. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is shown as a loading control. ( C ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by MDCK cells co-expressing HAI-1, as indicated. Densitometric analysis (average ± s.d. of 3 independent experiments, Figure 2—source data 2 ) shows the ratio between the short and the long uromodulin isoforms in the absence or presence of HAI-1 co-expression, as indicated. The serine protease inhibitor HAI-1 strongly reduces the amount of the short uromodulin isoform released in the culturing medium by MDCK cells. *p

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Expressing, Protease Inhibitor, Immunofluorescence, Western Blot, Transfection

    Model of uromodulin shedding and polymerisation. Uromodulin is exclusively expressed by TAL tubular epithelial cells. The protein enters the secretory pathway and reaches the plasma membrane in a polymerisation-incompetent conformation. This is ensured by the interaction between two hydrophobic patches within and next to the ZP-C subdomain (Internal Hydrophobic Patch, red circle, and External Hydrophobic Patch, dark green circle) ( Jovine et al., 2004 ; Schaeffer et al., 2009 ; Han et al., 2010 ). Shedding by hepsin at the uromodulin consensus cleavage site (red diamond), likely occurring at the plasma membrane, releases the hydrophobic interaction, generating polymerisation-competent species that are assembled into polymeric filaments within the tubular lumen. Pink circles indicate N-terminal EGF-like domains, yellow and green cylinders represent ZP-N and ZP-C subdomains. The orientation of uromodulin within polymers is hypothetical. DOI: http://dx.doi.org/10.7554/eLife.08887.025

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Model of uromodulin shedding and polymerisation. Uromodulin is exclusively expressed by TAL tubular epithelial cells. The protein enters the secretory pathway and reaches the plasma membrane in a polymerisation-incompetent conformation. This is ensured by the interaction between two hydrophobic patches within and next to the ZP-C subdomain (Internal Hydrophobic Patch, red circle, and External Hydrophobic Patch, dark green circle) ( Jovine et al., 2004 ; Schaeffer et al., 2009 ; Han et al., 2010 ). Shedding by hepsin at the uromodulin consensus cleavage site (red diamond), likely occurring at the plasma membrane, releases the hydrophobic interaction, generating polymerisation-competent species that are assembled into polymeric filaments within the tubular lumen. Pink circles indicate N-terminal EGF-like domains, yellow and green cylinders represent ZP-N and ZP-C subdomains. The orientation of uromodulin within polymers is hypothetical. DOI: http://dx.doi.org/10.7554/eLife.08887.025

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques:

    Mass spectrometry analysis of the long uromodulin isoform released by MDCK cells. ( A ) Mass spectrometry sequence coverage (42%) of Asp-N-digested human uromodulin (long isoform) from the medium of stably expressing MDCK cells. The analysis was carried out as described in the Methods section of the manuscript. The long isoform was separated in 2D electrophoresis. Matching peptides are shown in red, while the C- terminal peptide is shown in blue. This peptide ends at A611, a distal C-terminal relative to the one identified for the short isoform (F587) that is identical to the one reported for human urinary uromodulin (Santambrogio et al., 2008). ( B ) Representative tandem mass-spectrometry (MS/MS) spectrum confirming the identity of the 594 DQSRVLNLGPITRKGVQA 611 C-terminal peptide of the long uromodulin isoform released by MDCK cells and table of fragmented ions.

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Mass spectrometry analysis of the long uromodulin isoform released by MDCK cells. ( A ) Mass spectrometry sequence coverage (42%) of Asp-N-digested human uromodulin (long isoform) from the medium of stably expressing MDCK cells. The analysis was carried out as described in the Methods section of the manuscript. The long isoform was separated in 2D electrophoresis. Matching peptides are shown in red, while the C- terminal peptide is shown in blue. This peptide ends at A611, a distal C-terminal relative to the one identified for the short isoform (F587) that is identical to the one reported for human urinary uromodulin (Santambrogio et al., 2008). ( B ) Representative tandem mass-spectrometry (MS/MS) spectrum confirming the identity of the 594 DQSRVLNLGPITRKGVQA 611 C-terminal peptide of the long uromodulin isoform released by MDCK cells and table of fragmented ions.

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Mass Spectrometry, Sequencing, Stable Transfection, Expressing, Two-Dimensional Gel Electrophoresis

    Uromodulin secretion is not affected by lack of prostasin in vivo . ( A ) Transcript level of Prss8 , as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh ). Expression of Prss8 is detected in proximal convoluted tubules (PCT), proximal straight tubules (PST) and, to a lesser extent, in thick ascending limb (TAL) and collecting ducts (CD). Bars indicate average ± s.e.m. of 3 independent experiments ( Figure 7—source data 1 ). ( B ) Immunofluorescence analysis of mouse kidney sections shows strong signal of endogenous prostasin on the apical plasma membrane of proximal tubules, and weak signal on the apical plasma membrane of TAL epithelial cells where it co-localises with uromodulin. Scale bar, 20 µm. ( C ) Representative Western blot analysis of urinary uromodulin from control Prss8 lox/lox or Prss8 -/- mice. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows that uromodulin secretion is comparable between Prss8 -/- mice and control Prss8 lox/lox animals (average ± s.d., n = 5/group, Figure 7—source data 2 ) (Student’s t test). ( D ) Representative Western blot analysis of N-deglycosylated urinary uromodulin secreted by Prss8 -/- mice or control animals. An isoform of identical molecular weight, corresponding to the short uromodulin isoform, is detected in urine samples of both genotypes (n = 5/group). ( E ) Mass spectrometry sequence coverage (55% over the entire protein) of AspN-digested mouse uromodulin (UniProt accession Q91X17) purified from urine of Prss8 -/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at F588, the same C-terminal residue identified in urinary uromodulin of wild-type mice (Santambrogio et al., 2008) and control Prss8 lox/lox animals (data not shown). ( F ) Representative MS/MS spectrum confirming the sequence of urinary uromodulin C-terminal peptide ( 573 DSTSEQCKPTCSGTRF 588 ) in Prss8 -/- mice and table of fragmented ions. DOI: http://dx.doi.org/10.7554/eLife.08887.024

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Uromodulin secretion is not affected by lack of prostasin in vivo . ( A ) Transcript level of Prss8 , as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh ). Expression of Prss8 is detected in proximal convoluted tubules (PCT), proximal straight tubules (PST) and, to a lesser extent, in thick ascending limb (TAL) and collecting ducts (CD). Bars indicate average ± s.e.m. of 3 independent experiments ( Figure 7—source data 1 ). ( B ) Immunofluorescence analysis of mouse kidney sections shows strong signal of endogenous prostasin on the apical plasma membrane of proximal tubules, and weak signal on the apical plasma membrane of TAL epithelial cells where it co-localises with uromodulin. Scale bar, 20 µm. ( C ) Representative Western blot analysis of urinary uromodulin from control Prss8 lox/lox or Prss8 -/- mice. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows that uromodulin secretion is comparable between Prss8 -/- mice and control Prss8 lox/lox animals (average ± s.d., n = 5/group, Figure 7—source data 2 ) (Student’s t test). ( D ) Representative Western blot analysis of N-deglycosylated urinary uromodulin secreted by Prss8 -/- mice or control animals. An isoform of identical molecular weight, corresponding to the short uromodulin isoform, is detected in urine samples of both genotypes (n = 5/group). ( E ) Mass spectrometry sequence coverage (55% over the entire protein) of AspN-digested mouse uromodulin (UniProt accession Q91X17) purified from urine of Prss8 -/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at F588, the same C-terminal residue identified in urinary uromodulin of wild-type mice (Santambrogio et al., 2008) and control Prss8 lox/lox animals (data not shown). ( F ) Representative MS/MS spectrum confirming the sequence of urinary uromodulin C-terminal peptide ( 573 DSTSEQCKPTCSGTRF 588 ) in Prss8 -/- mice and table of fragmented ions. DOI: http://dx.doi.org/10.7554/eLife.08887.024

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: In Vivo, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Western Blot, Mouse Assay, Concentration Assay, Molecular Weight, Mass Spectrometry, Sequencing, Purification

    PIC treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: PIC treatment does not affect uromodulin intracellular distribution and expression. ( A ) Immunofluorescence analysis showing intracellular distribution of uromodulin in MDCK cells treated with vehicle (DMSO) (ctr) or protease inhibitor cocktail (PIC). KDEL is a marker of the endoplasmic reticulum (ER). Scale bar, 16 µm. ( B ) Representative Western blot analysis of uromodulin in lysates of MDCK cells under the same conditions as above. The upper band corresponds to the mature, fully glycosylated protein, the lower band corresponds to the immature protein carrying ER-type glycosylation (Schaeffer et al., 2012). Alpha-tubulin is shown as a loading control. PIC treatment does not alter uromodulin intracellular distribution nor its expression. DOI: http://dx.doi.org/10.7554/eLife.08887.007

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Expressing, Immunofluorescence, Protease Inhibitor, Marker, Western Blot

    Defective uromodulin urinary secretion in mice lacking hepsin. ( A ) Transcript level of Hpn , as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh ). Expression of Hpn is detected in proximal straight tubules (PST), thick ascending limb (TAL), proximal convoluted tubules (PCT), distal convoluted tubules (DCT) and, to a lesser extent, in collecting ducts (CD). Minimal expression of the protease is detected in glomeruli (GI). Bars indicate average ± s.e.m. of 3 independent experiments ( Figure 6—source data 1 ). ( B ) Immunofluorescence analysis of mouse kidney sections showing co-localisation of endogenous hepsin and uromodulin on the apical plasma membrane of TAL epithelial cells. Scale bar, 20 µm. ( C ) Representative Western blot analysis of urinary uromodulin secretion in Hpn -/- mice or control animals. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows reduced uromodulin urinary secretion in animals lacking hepsin (average ± s.d., n = 10/group, Figure 6—source data 2 ). ***p

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Defective uromodulin urinary secretion in mice lacking hepsin. ( A ) Transcript level of Hpn , as assessed by Real-Time qPCR on microdissected nephron segments (normalised to Gapdh ). Expression of Hpn is detected in proximal straight tubules (PST), thick ascending limb (TAL), proximal convoluted tubules (PCT), distal convoluted tubules (DCT) and, to a lesser extent, in collecting ducts (CD). Minimal expression of the protease is detected in glomeruli (GI). Bars indicate average ± s.e.m. of 3 independent experiments ( Figure 6—source data 1 ). ( B ) Immunofluorescence analysis of mouse kidney sections showing co-localisation of endogenous hepsin and uromodulin on the apical plasma membrane of TAL epithelial cells. Scale bar, 20 µm. ( C ) Representative Western blot analysis of urinary uromodulin secretion in Hpn -/- mice or control animals. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows reduced uromodulin urinary secretion in animals lacking hepsin (average ± s.d., n = 10/group, Figure 6—source data 2 ). ***p

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Western Blot, Concentration Assay

    Hepsin and prostasin directly cleave uromodulin in vitro . ( A ) Schematic representation of human uromodulin domain structure as shown in Figure 1A . The region not included in recombinant efUmod is shadowed. ( B ) The deletion of the elastase-sensitive fragment of uromodulin does not affect protein polymerisation on the surface of MDCK cells, as shown by immunofluorescence analysis (efUmod wt). As for full-length uromodulin ( Schaeffer et al., 2009 ), this process depends on correct protein cleavage at the physiological site, since it is abolished when the consensus cleavage site is mutated (efUmod 4Ala, carrying the mutation 586 RFRS 589 > 586 AAAA 589 ). Scale bar, 50 µm. ( C ) Purified efUmod, either wild-type (efUmod wt) or mutated at the consensus cleavage site (efUmod YAla, carrying the mutation 586 RFRS 589 > 586 AYAA 589 ), was incubated with recombinant prostasin or hepsin, as indicated. Both proteases decrease the mass of wild-type efUmod (white arrowheads in upper and middle panels) and cause the loss of its C-terminal His-tag (lower panel). Hepsin is more efficient than prostasin, as it drives complete digestion of the product, despite being used at 20x lower concentration (picomolar ratio between protease and efUmod was 1:100 for hepsin and 1:5 for prostasin, see lanes 7 and 8 for comparison). The asterisk indicates His-tagged prostasin. DOI: http://dx.doi.org/10.7554/eLife.08887.011

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Hepsin and prostasin directly cleave uromodulin in vitro . ( A ) Schematic representation of human uromodulin domain structure as shown in Figure 1A . The region not included in recombinant efUmod is shadowed. ( B ) The deletion of the elastase-sensitive fragment of uromodulin does not affect protein polymerisation on the surface of MDCK cells, as shown by immunofluorescence analysis (efUmod wt). As for full-length uromodulin ( Schaeffer et al., 2009 ), this process depends on correct protein cleavage at the physiological site, since it is abolished when the consensus cleavage site is mutated (efUmod 4Ala, carrying the mutation 586 RFRS 589 > 586 AAAA 589 ). Scale bar, 50 µm. ( C ) Purified efUmod, either wild-type (efUmod wt) or mutated at the consensus cleavage site (efUmod YAla, carrying the mutation 586 RFRS 589 > 586 AYAA 589 ), was incubated with recombinant prostasin or hepsin, as indicated. Both proteases decrease the mass of wild-type efUmod (white arrowheads in upper and middle panels) and cause the loss of its C-terminal His-tag (lower panel). Hepsin is more efficient than prostasin, as it drives complete digestion of the product, despite being used at 20x lower concentration (picomolar ratio between protease and efUmod was 1:100 for hepsin and 1:5 for prostasin, see lanes 7 and 8 for comparison). The asterisk indicates His-tagged prostasin. DOI: http://dx.doi.org/10.7554/eLife.08887.011

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: In Vitro, Recombinant, Immunofluorescence, Mutagenesis, Purification, Incubation, Concentration Assay

    Hepsin and prostasin expression in MDCK and HEK293 cells. ( A ) RT-PCR analysis showing gene expression of candidate proteases hepsin ( HPN ) and prostasin ( PRSS8 ) in MDCK and HEK293 cells. Constructs containing coding sequences of the human proteases were used as PCR positive controls (C+). Expression of GAPDH is shown as a cDNA positive control. PRSS8 and HPN are exclusively expressed in MDCK cells, confirming data obtained from available transcriptomes. ( B ) Representative Western blot analysis of uromodulin, hepsin and prostasin in lysates of transfected HEK293 cells. Wild-type proteases as well as catalytically inactive enzymes were expressed in HEK293 cells, as indicated. Protein disulfide-isomerase (Pdi) is shown as a loading control. The arrowhead points at hepsin specific band. DOI: http://dx.doi.org/10.7554/eLife.08887.010

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Hepsin and prostasin expression in MDCK and HEK293 cells. ( A ) RT-PCR analysis showing gene expression of candidate proteases hepsin ( HPN ) and prostasin ( PRSS8 ) in MDCK and HEK293 cells. Constructs containing coding sequences of the human proteases were used as PCR positive controls (C+). Expression of GAPDH is shown as a cDNA positive control. PRSS8 and HPN are exclusively expressed in MDCK cells, confirming data obtained from available transcriptomes. ( B ) Representative Western blot analysis of uromodulin, hepsin and prostasin in lysates of transfected HEK293 cells. Wild-type proteases as well as catalytically inactive enzymes were expressed in HEK293 cells, as indicated. Protein disulfide-isomerase (Pdi) is shown as a loading control. The arrowhead points at hepsin specific band. DOI: http://dx.doi.org/10.7554/eLife.08887.010

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Construct, Polymerase Chain Reaction, Positive Control, Western Blot, Transfection

    Urinary uromodulin misprocessing in Hpn -/- mice. ( A ) Mass spectrometry (MS) sequence coverage (51% over the entire protein) of trypsin-digested mouse uromodulin (long isoform) (UniProt accession Q91X17) purified from urine of Hpn -/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at K616, a distal C-terminal residue with respect to the one reported for mouse urinary uromodulin (F588 [Santambrogio et al., 2008]). ( B ) Representative tandem mass-spectrometry (MS/MS) spectrum, confirming the identity of the identified C-terminal peptide ( 608 QGVQASVSK 616 ) of the long uromodulin isoform released by Hpn -/- mice, and table of fragmented ions. DOI: http://dx.doi.org/10.7554/eLife.08887.023

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: Urinary uromodulin misprocessing in Hpn -/- mice. ( A ) Mass spectrometry (MS) sequence coverage (51% over the entire protein) of trypsin-digested mouse uromodulin (long isoform) (UniProt accession Q91X17) purified from urine of Hpn -/- mice. Matching peptides are shown in red, while the C-terminal peptide is shown in blue. This peptide ends at K616, a distal C-terminal residue with respect to the one reported for mouse urinary uromodulin (F588 [Santambrogio et al., 2008]). ( B ) Representative tandem mass-spectrometry (MS/MS) spectrum, confirming the identity of the identified C-terminal peptide ( 608 QGVQASVSK 616 ) of the long uromodulin isoform released by Hpn -/- mice, and table of fragmented ions. DOI: http://dx.doi.org/10.7554/eLife.08887.023

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Mouse Assay, Mass Spectrometry, Sequencing, Purification

    MDCK cells as a model to study physiological uromodulin shedding. ( A ) Schematic representation of human uromodulin domain structure containing a leader peptide (predicted to be cleaved at residue 23), three EGF-like domains, a central domain with 8 conserved cysteines (D8C), a bipartite Zona Pellucida (ZP) domain (ZP-N/ZP-C) and a glycosylphosphatidylinositol (GPI)-anchoring site (predicted at position 614). Internal (IHP) and External (EHP) Hydrophobic Patches ( Jovine et al., 2004 ; Schaeffer et al., 2009 ), Consensus Cleavage Site (CCS) and seven N-glycosylation sites (Ψ) are also indicated. ( B ) Immunofluorescence analysis of non-permeabilised MDCK cells expressing uromodulin. Polymers formed by the protein are clearly detected on the cell surface. Scale bar, 50 µm. ( C ) Electron microscopy analysis of uromodulin polymers purified from the medium of MDCK cells. The arrows indicate the typical protrusions of uromodulin filaments spaced about 130 Å. Scale bar, 100 nm. ( D ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by transfected MDCK cells or purified from urine. A single isoform is clearly seen in the urinary sample. An isoform with similar molecular weight is released by MDCK cells (white arrowhead), which also secrete a longer and more abundant one (black arrowhead). ( E ) Representative tandem mass-spectrometry (MS/MS) spectrum confirming the identity of the C-terminal peptide 572 DTMNEKCKPTCSGTRF 587 of the short uromodulin isoform released by MDCK cells and table of fragmented ions. The C-terminal residue F587 is identical to the one that we mapped in human urinary protein ( Santambrogio et al., 2008 ). DOI: http://dx.doi.org/10.7554/eLife.08887.003

    Journal: eLife

    Article Title: The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin

    doi: 10.7554/eLife.08887

    Figure Lengend Snippet: MDCK cells as a model to study physiological uromodulin shedding. ( A ) Schematic representation of human uromodulin domain structure containing a leader peptide (predicted to be cleaved at residue 23), three EGF-like domains, a central domain with 8 conserved cysteines (D8C), a bipartite Zona Pellucida (ZP) domain (ZP-N/ZP-C) and a glycosylphosphatidylinositol (GPI)-anchoring site (predicted at position 614). Internal (IHP) and External (EHP) Hydrophobic Patches ( Jovine et al., 2004 ; Schaeffer et al., 2009 ), Consensus Cleavage Site (CCS) and seven N-glycosylation sites (Ψ) are also indicated. ( B ) Immunofluorescence analysis of non-permeabilised MDCK cells expressing uromodulin. Polymers formed by the protein are clearly detected on the cell surface. Scale bar, 50 µm. ( C ) Electron microscopy analysis of uromodulin polymers purified from the medium of MDCK cells. The arrows indicate the typical protrusions of uromodulin filaments spaced about 130 Å. Scale bar, 100 nm. ( D ) Representative Western blot analysis of N-deglycosylated uromodulin secreted by transfected MDCK cells or purified from urine. A single isoform is clearly seen in the urinary sample. An isoform with similar molecular weight is released by MDCK cells (white arrowhead), which also secrete a longer and more abundant one (black arrowhead). ( E ) Representative tandem mass-spectrometry (MS/MS) spectrum confirming the identity of the C-terminal peptide 572 DTMNEKCKPTCSGTRF 587 of the short uromodulin isoform released by MDCK cells and table of fragmented ions. The C-terminal residue F587 is identical to the one that we mapped in human urinary protein ( Santambrogio et al., 2008 ). DOI: http://dx.doi.org/10.7554/eLife.08887.003

    Article Snippet: Antibodies We used the following primary antibodies: mouse anti-HA (MMS-101P, 1:1000 for WB and 1:500 for IF, Covance), rabbit anti-FLAG (F7425, 1:500 for IF, Sigma-Aldrich), goat anti-Myc (NB600-335, 1:500 for IF, Novus Biologicals, Littleton, CO), sheep anti-uromodulin (T0850, 1:1000 for WB, US Biological, Salem, MA), sheep anti-uromodulin (K90071C, 1:200 for IF, Meridian Life Science, Cincinnati, OH), goat anti-uromodulin (55140, 1:1000 for WB and 1:500 for IF, MP Biomedicals, Santa Ana, CA), rabbit anti-hepsin (100022, 1:1000 for WB and 1:50 for IF, Cayman Chemical), sheep anti-prostasin (AF4599, 1:1000 for WB and 1:200 for IF, R & D System, Minneapolis, MN), rabbit anti-prostasin (kind gift of Prof. Carl Chai, University of Central Florida College of Medicine, FL; 1:200 for IF) , rabbit anti-HAI-1 (H-180, 1:1000 for WB, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PDI (H-160, 1:1000 for WB, Santa Cruz Biotechnology), mouse anti-E-cadherin (610182, 1:500 for IF, BD Biosciences, San Jose, CA), mouse anti-KDEL (ADI-SPA-827-D, 1:500, Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (6C5, 1:5000 for WB, Santa Cruz Biotechnology), mouse anti-β actin (A2228, 1:16000 for WB, Sigma-Aldrich), mouse anti-α tubulin (SC-8035, 1:1000 for WB, Santa Cruz Biotechnology) and mouse anti-5His (34660, 1:1000 for WB, Qiagen, Venlo, The Netherlands).

    Techniques: Immunofluorescence, Expressing, Electron Microscopy, Purification, Western Blot, Transfection, Molecular Weight, Mass Spectrometry

    Schematic of plant expression plasmids pPHAP1301-HRVVP7-CTB and pPHAP1301-HRVVP7. bar encodes a resistance marker for glufosinate selection. (A) HRVVP7 coding sequence. (Gly 4 Ser) 3 is the linker between HRVVP7 and CTB . (B) HRVVP7 coding sequence. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; Phas , β-phaseolin storage protein; 35s, Cauliflower mosaic virus; LB, left border; RB, right border; nos , nopaline synthase.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Oral immunization with rotavirus VP7-CTB fusion expressed in transgenic Arabidopsis thaliana induces antigen-specific IgA and IgG and passive protection in mice

    doi: 10.3892/etm.2018.6003

    Figure Lengend Snippet: Schematic of plant expression plasmids pPHAP1301-HRVVP7-CTB and pPHAP1301-HRVVP7. bar encodes a resistance marker for glufosinate selection. (A) HRVVP7 coding sequence. (Gly 4 Ser) 3 is the linker between HRVVP7 and CTB . (B) HRVVP7 coding sequence. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; Phas , β-phaseolin storage protein; 35s, Cauliflower mosaic virus; LB, left border; RB, right border; nos , nopaline synthase.

    Article Snippet: The intracellular rotavirus was detected using the mouse mAb directed against rotavirus VP7 (cat. no. C01715M; Meridian Life Science, Inc., Memphis, TN, USA; 1:500 dilution) as the primary antibody at 25°C for 30 min and fluorescein-isothiocyanate-labeled goat anti-mouse IgG antibody (cat. no. ab6785; Abcam) as the secondary antibody (1:500 dilution) at 37°C for 1 h. The cells were washed three times with PBS at room temperature (15 min/wash).

    Techniques: Expressing, CtB Assay, Marker, Selection, Sequencing

    HRVVP7-CTB molecular detection was performed using polymerase chain reaction and western blotting. Agarose gel electrophoresis was used to screen for positive transgenic lines following amplification. (A) HRVVP7-CTB fusion transgenic Arabidopsis thaliana lines. pUC19-HRVVP7-linker-CTB plasmid was used as the positive control (+). (B) HRVVP7 transgenic A. thaliana lines. pPHAP1301-HRVVP7 plasmid was used as the positive control (+). Proteins expressed by transgenic A. thaliana were assessed using western blotting. (C) HRVVP7-linker-CTB and (D) HRVVP7 expressed in seeds of T3 transgenic A. thaliana lines. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; WT, wild type A. thaliana; +, positive control; M, marker.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Oral immunization with rotavirus VP7-CTB fusion expressed in transgenic Arabidopsis thaliana induces antigen-specific IgA and IgG and passive protection in mice

    doi: 10.3892/etm.2018.6003

    Figure Lengend Snippet: HRVVP7-CTB molecular detection was performed using polymerase chain reaction and western blotting. Agarose gel electrophoresis was used to screen for positive transgenic lines following amplification. (A) HRVVP7-CTB fusion transgenic Arabidopsis thaliana lines. pUC19-HRVVP7-linker-CTB plasmid was used as the positive control (+). (B) HRVVP7 transgenic A. thaliana lines. pPHAP1301-HRVVP7 plasmid was used as the positive control (+). Proteins expressed by transgenic A. thaliana were assessed using western blotting. (C) HRVVP7-linker-CTB and (D) HRVVP7 expressed in seeds of T3 transgenic A. thaliana lines. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; WT, wild type A. thaliana; +, positive control; M, marker.

    Article Snippet: The intracellular rotavirus was detected using the mouse mAb directed against rotavirus VP7 (cat. no. C01715M; Meridian Life Science, Inc., Memphis, TN, USA; 1:500 dilution) as the primary antibody at 25°C for 30 min and fluorescein-isothiocyanate-labeled goat anti-mouse IgG antibody (cat. no. ab6785; Abcam) as the secondary antibody (1:500 dilution) at 37°C for 1 h. The cells were washed three times with PBS at room temperature (15 min/wash).

    Techniques: CtB Assay, Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis, Transgenic Assay, Amplification, Plasmid Preparation, Positive Control, Marker

    Schematic of plant expression plasmids pPHAP1301-HRVVP7-CTB and pPHAP1301-HRVVP7. bar encodes a resistance marker for glufosinate selection. (A) HRVVP7 coding sequence. (Gly 4 Ser) 3 is the linker between HRVVP7 and CTB . (B) HRVVP7 coding sequence. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; Phas , β-phaseolin storage protein; 35s, Cauliflower mosaic virus; LB, left border; RB, right border; nos , nopaline synthase.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Oral immunization with rotavirus VP7-CTB fusion expressed in transgenic Arabidopsis thaliana induces antigen-specific IgA and IgG and passive protection in mice

    doi: 10.3892/etm.2018.6003

    Figure Lengend Snippet: Schematic of plant expression plasmids pPHAP1301-HRVVP7-CTB and pPHAP1301-HRVVP7. bar encodes a resistance marker for glufosinate selection. (A) HRVVP7 coding sequence. (Gly 4 Ser) 3 is the linker between HRVVP7 and CTB . (B) HRVVP7 coding sequence. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; Phas , β-phaseolin storage protein; 35s, Cauliflower mosaic virus; LB, left border; RB, right border; nos , nopaline synthase.

    Article Snippet: The intracellular rotavirus was detected using the mouse mAb directed against rotavirus VP7 (cat. no. C01715M; Meridian Life Science, Inc., Memphis, TN, USA; 1:500 dilution) as the primary antibody at 25°C for 30 min and fluorescein-isothiocyanate-labeled goat anti-mouse IgG antibody (cat. no. ab6785; Abcam) as the secondary antibody (1:500 dilution) at 37°C for 1 h. The cells were washed three times with PBS at room temperature (15 min/wash).

    Techniques: Expressing, CtB Assay, Marker, Selection, Sequencing

    HRVVP7-CTB molecular detection was performed using polymerase chain reaction and western blotting. Agarose gel electrophoresis was used to screen for positive transgenic lines following amplification. (A) HRVVP7-CTB fusion transgenic Arabidopsis thaliana lines. pUC19-HRVVP7-linker-CTB plasmid was used as the positive control (+). (B) HRVVP7 transgenic A. thaliana lines. pPHAP1301-HRVVP7 plasmid was used as the positive control (+). Proteins expressed by transgenic A. thaliana were assessed using western blotting. (C) HRVVP7-linker-CTB and (D) HRVVP7 expressed in seeds of T3 transgenic A. thaliana lines. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; WT, wild type A. thaliana; +, positive control; M, marker.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Oral immunization with rotavirus VP7-CTB fusion expressed in transgenic Arabidopsis thaliana induces antigen-specific IgA and IgG and passive protection in mice

    doi: 10.3892/etm.2018.6003

    Figure Lengend Snippet: HRVVP7-CTB molecular detection was performed using polymerase chain reaction and western blotting. Agarose gel electrophoresis was used to screen for positive transgenic lines following amplification. (A) HRVVP7-CTB fusion transgenic Arabidopsis thaliana lines. pUC19-HRVVP7-linker-CTB plasmid was used as the positive control (+). (B) HRVVP7 transgenic A. thaliana lines. pPHAP1301-HRVVP7 plasmid was used as the positive control (+). Proteins expressed by transgenic A. thaliana were assessed using western blotting. (C) HRVVP7-linker-CTB and (D) HRVVP7 expressed in seeds of T3 transgenic A. thaliana lines. HRVVP7, human rotavirus VP7; CTB, cholera toxin B subunit; WT, wild type A. thaliana; +, positive control; M, marker.

    Article Snippet: The intracellular rotavirus was detected using the mouse mAb directed against rotavirus VP7 (cat. no. C01715M; Meridian Life Science, Inc., Memphis, TN, USA; 1:500 dilution) as the primary antibody at 25°C for 30 min and fluorescein-isothiocyanate-labeled goat anti-mouse IgG antibody (cat. no. ab6785; Abcam) as the secondary antibody (1:500 dilution) at 37°C for 1 h. The cells were washed three times with PBS at room temperature (15 min/wash).

    Techniques: CtB Assay, Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis, Transgenic Assay, Amplification, Plasmid Preparation, Positive Control, Marker