To further define the cleavage site of epithelial cell proteases, the intact mass of C-terminal fragments generated by either the cleavage of C2 fragment or the cleavage of NHBA protein was measured by mass spectrometry. were recognized with DAPI (blue staining). No significant changes in ZO-1 distribution were observed indicating that TJs integrity was maintained.(TIF) pone.0194662.s001.tif (1.2M) GUID:?A20A8FBB-F6AC-461D-8823-00EAFC907279 S2 Fig: C2 fragment and NHBA protein are processed by cell proteases secreted by differentiated NHBE epithelial cells. Western blot analysis of recombinant C2 fragment or NHBA full-length protein incubated with cell supernatants prepared from differentiated NHBE cells. Samples were analyzed at different time points (45, 1h, 2h and 4h). Polyclonal mouse sera against C2 fragment (A) or polyclonal mouse sera against NHBA full-length protein (B) were utilized for blotting the membranes. The arrow shows the recombinant C2 fragment. The arrowhead shows the Naspm recombinant NHBA full-length protein. The asterisk and the open arrowhead shows the C-terminal fragment and the N-terminal fragment, respectively, derived from the cleavage of epithelial cell proteases.(TIF) pone.0194662.s002.tif (3.0M) GUID:?5F5846D0-2A7F-4669-8A18-396540AD595A S3 Fig: mRR NHBA mutant protein is not cleaved by epithelial cell proteases. Western blot analysis of supernatants of polarized Calu-3 cells treated with 5 M of recombinant mRR NHBA mutant protein. Samples were collected at different time points (45, 2h, 4h and 24h). Polyclonal mouse sera against NHBA full-length protein were utilized for blotting the membrane. Recombinant NHBA C-terminal fragments, C2 and C1, were loaded as settings for the blotting.(TIF) pone.0194662.s003.tif (2.0M) GUID:?909DC16C-02D1-4520-85AF-DB2E65AC012C S4 Fig: Identification of cell supernatant fractions enriched with the epithelial cell protease responsible for NHBA cleavage. Ion exchange chromatography of polarized Calu-3 cell supernatant A) Chromatogram Naspm shows elution of fractions (in reddish), protein absorbance at 280nm (in blue), salt concentration (in green) and conductivity (in brownish). B and C) SDS-PAGE analysis of each eluted portion incubated over night (o/n) with 5 M of recombinant C2-fragment (B) or with 5 M of recombinant NHBA full size protein (C). Proteins were stained with blue coomassie.(TIF) pone.0194662.s004.tif (987K) GUID:?B8D69D6A-C4B0-4FAB-906B-307A8179E830 S5 Fig: Screening of protease inhibitors. Western blot analysis of recombinant C2 fragment (A) or NHBA full-length protein (B) incubated for 2 hours with Calu-3 cell supernatants that were pre-treated or not with protease inhibitors for 30 minutes. Protease inhibitors tested: EDTA, Leupeptin (Leu), Pepstatine A (Pep), GI254023X and E-64. Polyclonal mouse sera against C2 fragment (A) or against NHBA full-length protein (B) were utilized for blotting the membranes.(TIF) pone.0194662.s005.tif (2.1M) GUID:?034A87A3-F121-4616-B098-FFD88FB0043C S6 Fig: Calu-3 epithelial cells express complement component C3 and factor B. Agarose gel electrophoresis analysis of manifestation of human being CFB and C3 genes in Calu-3 cells (A) and polarized Calu-3 cells (B). GAPDH was used as internal positive control. Total RNA was isolated from epithelial cells, retro Naspm transcribed with oligo(dT) and cDNA were used as themes for PCR amplification. For each gene analyzed, specific oligonucleotides amplified part of the mRNA (GAPDH: 518 nt; CFB: 885 nt; C3: 408 nt).(TIF) pone.0194662.s006.tif (1.3M) GUID:?C2C9ADB0-4E9B-446E-8172-26C1FC421B68 S7 Fig: EDTA does not inhibit the activity of kallikrein. A) SDS-PAGE analysis of recombinant C2 fragment incubated over night AF6 with plasma-purified kallikrein pre-treated or not with EDTA. Proteins were stained with blue coomassie. B) Western blot analysis of recombinant NHBA full-length protein incubated over night with plasma-purified kallikrein pre-treated or not with EDTA. Polyclonal mouse sera against NHBA full-length protein were utilized for blotting the membrane.(TIF) pone.0194662.s007.tif (1.6M) GUID:?AF307443-49B1-4FB9-825A-A15241DEBC3E S1 Table: List of proteins contained in the determined fractions of polarized Calu-3 cell supernatant recognized by mass spectrometry. (PDF) pone.0194662.s008.pdf (573K) GUID:?74F175E0-0A00-449C-A150-3BDD0922A484 S1 Text:.