The HBEC mucosal surfaces were washed with 500 ��l PBS for 30 min prior to experimentation to remove selleckchem accumulated endogenous SPLUNC1. SPLUNC1 recovery into the ASL was measured over time by lavaging with 200 ��l PBS at timed intervals after the initial wash/volume load. To detect SPLUNC1, 20% by volume of the lavage was transferred onto a nitrocellulose membrane using a slot-blot apparatus (GE Healthcare, Pittsburgh, PA, USA). The blot was processed as described above using an anti-hPLUNC primary antibody (R&D Systems, Minneapolis, MN, USA). Transepithelial potential difference (Vt) studies A single-barreled Vt-sensing electrode was positioned in the ASL by micromanipulator and used in conjunction with a macroelectrode in the serosal solution to measure Vt using a voltmeter (World Precision Instruments, Sarasota, FL, USA), as described previously (16).

Circular dichroism (CD) G22-A39 (100 ��M) was dissolved in a buffer containing 10 mM sodium phosphate (pH 7.4) in a 1 mm cuvette. Using a Chirascan-plus instrument (Applied Photophysics Ltd., Leatherhead, UK), 5 individual spectra from 185 to 280 nm were recorded at 25 �� 1.0��C and averaged. All measurements were corrected for buffer signal. Expression and purification of human SPLUNC1 The SPLUNC1-��19 construct was created by cloning amino acid residues 20�C256 out of the SPLUNC1 cDNA described above for entry into pMCSG7 for protein expression. BL21-CodonPlus competent cells (Agilent Technologies, Santa Clara, CA, USA) were transformed with the expression plasmid and cultured in the presence of antifoam (50 ��l), ampicillin (100 ��g/ml), and chloramphenicol (34 ��g/ml) in Luria broth medium with vigorous shaking at 37��C until an OD600 of 0.

6 was attained. Expression was induced with the addition of isopropyl-1-thio-d-galactopyranoside (IPTG). Bacteria were collected by centrifugation at 4500 g for 20 min at 4��C. Cell pellets were resuspended in buffer A (20 mM potassium phosphate, pH 7.4; 50 mM imidazole; 500 mM NaCl; and 0.02% NaAzide) along with lysozyme, DNase1, and protease inhibitor tablets. Cells were sonicated, and cell lysate was separated into soluble and insoluble fractions using high-speed centrifugation. The soluble fraction was filtered, then flowed over a Ni-NTA His-Trap gravity column and washed with buffer A. The bound protein was eluted with buffer B (20 mM potassium phosphate, pH 7.

4; 500 mM imidazole; 500 mM NaCl; and 0.02% NaAzide) and separated using an S200 gel filtration column on an ?KTAxpress (GE Healthcare). The histidine tag was removed with Tev protease, leaving amino acid residues serine, GSK-3 asparagine, and alanine on the N terminus of the protein. Another round of nickel and HPLC purification removed the tag from the purified SPLUNC1-��19. We confirmed that SPLUNC1-��19 purified in this fashion inhibited ENaC HBECs (n=6).