Vuagniaux G, Vallet V, Jaeger NF, Hummler E, Rossier BC.

	Figure 1. . mCAP1, 2, and 3 are membrane-bound serine proteases. (A) Structural properties of the three mCAPs (mCAP1, mCAP2, and mCAP3). The amino acid sequence of each protein was scanned using ProfileScan algorithm to confirm the presence of domains indicated. Numbers delineate the location of each domain. (B) A representative phylogenetic tree of the GPI-anchored mCAP1 and type II transmembrane mCAP2 and mCAP3 serine protease family proteins. Multiple alignment was generated by the clustal W program (mCAP1, AAG1705; hProstasin, Q16651; xCAP1, AAB969054; mCAP2, AY043240; hTMPRSS4-1, CAC60389; hTMPRSS4–2; MT-SP2, Q9NRS4; mTMPRSS2; epitheliasin, AAF97867; hTMPRSS2, AAC51784; mTMPRSS3, CAC83350; hTMPRSS3, P57727; mCAP3; epithin, AAD02230; hMT-SP1, AAF00109; xMT-SP1, BAB08218). (C) Amino acid alignment of the serine protease catalytic domain of mCAP1, mCAP2, and mCAP3 with mouse trypsinogen precursor (1–246), was performed using Pileup program (Genetic Computer Group). Gray and black boxes indicate similar and identical residues, respectively. The catalytic triad (H, D, and S, asterisks), the amino acids implicated in the P1 substrate specificity (closed circle), and the position for Na+ sensibility (open circle) are indicated.
	Figure 2. . Coexpression of all three serine proteases with ENaC. Multiple tissue Northern blot containing 20 μg of RNA/lane were hybridized with cDNA fragments of the rat αENaC, mouse CAP1, CAP2, CAP3, and GAPDH genes, as described in materials and methods. mRNA transcript lengths are indicated.
	Figure 3. . Functional analysis of mCAP1–3 in Xenopus oocytes. (A) Comparison of the effect of mCAP1, mCAP2, and mCAP3 on INa in Xenopus oocytes. Oocytes were injected with rat ENaC subunits in the presence of either water (open bar, lane 1) or increasing amounts (2, 4, and 8 or 12 ng) of mCAP1 (closed bar, lanes 2–4), mCAP2 (light gray bar, lanes 5–8), or mCAP3 (dark gray bars, lanes 9–12). §§, P < 0.01 versus conditions with other amounts of the same cRNA protease. n ≥ 12 measured oocytes. (B) Effect of preincubation with aprotinin (lanes 3, 4, 7, 8, 11, 12, 15, 16) or perfusion of trypsin (gray bars, lanes 2, 4, 6, 8, 10, 12, 14, 16) on oocytes injected with either rENaC and water (lane 1–4) or rENaC together with 4 ng mCAP1 (lane 5–8), 8 ng mCAP2 (lane 9–12), and 2 ng mCAP3 (lane 13–16). n ≥ 12 measured oocytes.
	Figure 4. . Synergistic activation of mCAP1–3 and Sgk1 on ENaC activity. (A) Oocytes were injected with cRNA coding either for α, β, and γFLAG-tagged rENaC subunits (rENaCf) and water (lane 1) or rENaCf together with mCAP1 (lane 2), mCAP2 (lane 3), mCAP3 (lane 4), Sgk1 (lanes 5–8), or both Sgk1 and mCAP1 (lane 6), Sgk1 and mCAP2 (lane 7), and Sgk1 and mCAP3 (lane 8). INa was measured in absence (open bar) and after perfusion of trypsin (closed bar) and normalized to INa in oocytes injected with rENaC alone. n ≥ 15 measured oocytes per experimental condition taken from Tables I–III. (B) Normalized oocyte cell surface expression of rENaCf in oocytes experiments described in A. Water, lane 1; mCAP1, lane 2; mCAP2, lane 3; mCAP3, lane 4; Sgk1, lane 5; mCAP1 + Sgk1, lane 6; mCAP2 + Sgk1, lane 7; mCAP3 + Sgk1, lane 8. ***, P < 0.001 versus rENaCf + water.
	Figure 5. . Model of CAPs and Sgk1 action on ENaC. (A) CAPs increase the open probability of ENaC channels. The substrate of CAPs may be ENaC or protein(s) associated with ENaC. (B) Model of Sgk1 action on ENaC. Sgk1 principally increases the number of active channels at the cell surface by diminishing the removal of the channels from the cell surface and by increasing its Po. (C) CAPs proteases and Sgk1 act synergically on the channel open probability and cell surface expression.

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