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