The HBEC mucosal surfaces were washed with 500 ��l PBS for 30 min prior to experimentation to remove Vandetanib mechanism 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, Anacetrapib 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).