reduced with 10 mM dithiothreitol, alkylated with 20 mM iodoacetamide and digested with trypsin in the presence of 0.1% w/v RapiGest SF surfactant (Waters). The sample was spiked with 50 fmol of yeast alcohol dehydrogenase digest (Waters Mass Prep) per mg of total AH protein. One mg of protein from the digested AH was injected onto a nanoAcquity LC system, coupled to a Synapt HDMS mass spectrometer. The sample was analyzed in triplicate using MSE (data-independent acquisition) for quantita- tive/qualitative analysis, followed by a single data-dependent analysis (DDA) for supplementary identifications. Data were searched against the Swissprot Human (v57.5) database, with 20 ppm precursor and 0.04 Da product ion mass accuracy and trypsin specificity, with either PLGS 2.4/IdentityE (MSE) or Mascot v2.2 (DDA) search engine. Data was loaded into Scaffold 2 Proteome Software version 2.06 to generate a list of the most abundant proteins detected by mass spectrometry. Raw data alignment, feature detection, and label-free quantitation were performed within Rosetta Elucidator v3.3 software. The fmol quantity of each protein (per mg injected) was performed according to the method of Silva et al. [45] for the 89 proteins detected based on 2 or more peptides using yeast alcohol dehydrogenase as an internal standard. These analyses were performed at the Duke Proteomics Core Facility. TM cell cultures Human primary TM cells were cultured from fresh corneal rings donated by the Duke Ophthalmology Clinical Service after they were used for corneal transplantation as described previously [22]. Porcine primary TM cells were isolated and cultured using collagen IV digestion as described earlier by us [46]. Cell passages between 4 and 6 were used in this study. LysoPLD assay LysoPLD enzyme activity was assayed by measuring hydrolysis of lysophosphatidylcholine to choline and lysophosphatidic acid as described by Gijsbers et al. [27]. Briefly, choline derived from the hydrolysis of lysophosphatidylcholine was coupled with choline oxidase to oxidize choline into betaine and H2O2. Subsequently, the H2O2 generated reacts with Amplex Red reagent in the presence of horseradish peroxidase to generate resorufin, which is measured using a fluorescence spectrometer with excitation and emission filters at 530 nm and 590 nm, respectively. The original PLD assay was modified for lysoPLD by replacing the substrate phosphatidylcholine with lysophosphatidylcholine. The enzyme reactions were performed in duplicate in a final volume of 200 ml at 37uC for 30 minutes using a 96 well microtiter plates and Molecular Probe Spectramax M3. H2O2 standards, heat inacti- vated AH control and enzyme assays lacking lysophosphatidyl- choline substrate were run simultaneously. Enzyme activity was expressed as mM H2O2 generated per mg of total AH protein. Immunofluorescence TM cells were plated on glass coverslips coated with 2% gelatin and grown till they were 80% confluent. After treatments in serum-free media, they were washed twice with 16 phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized in 0.5% Triton-X 100 for 15 minutes, and blocked in serum buffer containing 10% FBS. Following this, cells were incubated with respective primary and Alexa fluor-conjugated secondary anti- bodies as we described earlier [22]. Finally, coverslips were washed and mounted onto glass slides with Aqua Mount (Lerner Laboratories, Pittsburg, PA) and observed under a Nikon confocal system (C1 Digital Eclipse). Autotaxin and IOP Immunoblotting Total cell lysates were prepared from confluent serum-starved TM cells. Briefly, cells were washed twice with cold PBS, and collected into lysis buffer containing 10 mM Tris, pH 7.4, 0.2 mM MgCl2, 5 mM N-ethylmaleimide, 0.4 mM iodoacetamide and 60 mM PMSF and protease inhibitor (cocktail tablets complete, Mini, EDTA-free, Roche, Basel, Switzerland), and phosphatase inhibitors (Phosphostop, Basel, Switzerland), sonicated on ice (probe sonicator, followed by bath sonicator for 30 min) and centrifuged at 800 g, 4uC for 15 min. The supernatant was collected and protein was measured as described below. Media was collected and centrifuged at 3000 rpm for 10 min at 4uC to remove cellular debris. The supernatant was concentrated using Millipore Centrifugal Filter Units with ,30 kDa cutoff. The filters were centrifuged at 2500 rpm at 4uC. Bio-Rad dye protein assay reagent was used to estimate the protein concentrations in both cell lysate and media samples using the Bradford method. Equal amounts of protein were dissolved in Laemmli buffer, boiled for 5 minutes, and separated by SDS-PAGE using 8% acrylamide gels. Proteins were transferred to nitrocellulose membrane, which were then incubated with LysoPLD polyclonal antibody (diluted 1:250 in TBST) overnight at 4uC. Immunoblots were developed using appropriate HRP (horse radish peroxidase)-conjugated secondary antibody and enhanced chemiluminescence, as we described earlier [22]. Densitometry of scanned films was performed using Adobe Photoshop CS3 Software, and results expressed relative to the loading control (b-tubulin). Myosin Light Chain (MLC) Phosphorylation Changes in TM cell myosin light chain phosphorylation status were determined by immunoblot analysis using a phospho-MLC specific antibody and urea-glycerol gel electrophoresis as we described earlier [15]. Densitometry of scanned films was performed using Adobe Photoshop CS3 Software, and results expressed relative to the loading control (b-tubulin). RT-PCR Total RNA was extracted using RNeasy Micro kit (Qiagen, Valencia, CA) per the user manual from P4 confluent cultures of HTM cells derived from human donors aged 33, 54 and 75 years. 2 mg of total RNA was used for first-strand synthesis using Superscript first-strand synthesis system for RT-PCR (Life Technologies, Carlsbad, CA). The resultant cDNA was used to amplify DNA specific to ATX isoforms in a total volume of 50 ml with 1.5 mM MgCl2, 0.2 mM dNTPs, 2 U Taq polymerase (Life Technologies), and 0.5 mM sense and antisense primers for the a, b, and c variants of ATX. GAPDH primers were used as positive control. The primer sequences (forward/reverse) used to amplify the different isoforms of ATX are listed below: 1. a – ATGGATTACAGCCACCAAGC/TCTTCCATTC- CATGGTCTCC 2. b - T C G G C C C T G A G A T G A C A A A T C / GAGGTGTCTCTCTTCTGTAG 3. c – A T G T C C G T G T T T C T C C G A G T / A T T G - CAGCTCTCCTCGTTGT 4. GAPDH – TGCACCACCAACTGCTTAGC/GGCATG- GACTGTGGTCATGAG. The amplification was carried out using GeneAmp Amplifica- tion System (Applied Biosciences) with a denaturation step at 94uC for 4 min, followed by 94uC for 1 min, annealing at 57uC for 60 s, and extension at 72uC for 60 s. This cycle was repeated 30 times PLOS ONE | www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e42627