(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 Prss8lox/loxor Prss8-/- mice. Urinary protein loading was normalised to urinary creatinine concentration. Densitometric analysis shows that uromodulin secretion is comparable between Prss8-/- mice and control Prss8lox/loxanimals (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 Prss8lox/loxanimals (data not shown). (F) Representative MS/MS spectrum confirming the sequence of urinary uromodulin C-terminal peptide (573DSTSEQCKPTCSGTRF588) in Prss8-/- mice and table of fragmented ions.