Autotaxin and IOP Figure 10. Cyclic mechanical stretch of TM cells induces ATX extracelluar and intracellular protein. A. Confluent cultures of primary porcine TM cells maintained under serum free condition and subjected to 10% cyclical mechanical stretch (one cycle/second) for 17 hrs revealed increased levels of ATX protein levels in the conditioned media as compared to the media derived from the un-stretched control cells based on immunoblot analysis with equal amount (10 mg) of total protein. For a loading control, SDS-PAGE separated proteins were stained with Coomassie blue and the staining intensity was compared (not shown). B. Densitometric semi-quantitative analysis of the immunoblots shown in panel A. *P,0.05, n = 4. C. Cell lysates derived from TM cells subjected to the cyclic mechanical stretch revealed increased levels of ATX protein compared to the samples of unstretched controls based on immunoblot analysis of equal amounts of protein (20 mg). b-tubulin was immunoblotted as a loading control. D. Densitometric semi-quantitative analysis of ATX immunoblots of cell lysates shown in panel C. * P,0.05, n = 4. doi:10.1371/journal.pone.0042627.g010 Informed written patient consent was obtained prior to sample collection and the principles expressed in the Declaration of Helsinki were followed. This study does not involve minors or children. Reagents RNeasy Mini kit (Qiagen, Valencia, CA), Advantage RT for PCR kit and Advantage cDNA PCR kit (BD Biosciences Clontech, Palo Alto, CA) were procured from the respective commercial sources. Mouse monoclonal anti-tubulin antibodies and Tetra- methyl rhodamine isothiocyanate (TRITC)–conjugated phalloidin were from Sigma-Aldrich (St. Louis, MO). Polyclonal phospho- MLC antibody (C.No. 36745) was from Cell Signalling Technology (Danvers, MA). L-a-Lysophosphatidylcholine (C.No. 830071P) was from Avanti Polar Lipids, Inc. (Alabaster, AL). ATX polyclonal antibody (C. No. 10005375) and its blocking peptide (C. No. 10007193) were obtained from Cayman Chemicals (Ann Arbor, MI), and S32826 (ATX inhibitor) were from Cayman Chemicals (C. No. 13664) and Sigma-Aldrich (C. No. S1825). Amplex Red PLD Assay Kit (C.No. A12219) was from Molecular Probes/Invitrogen (Grand Island, NY). Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) with low glucose were obtained from Gibco-BRL (Gaithersburg, MD). Bio- Rad protein assay reagent (C. No. 500-0006) was purchased from Bio-Rad. FITC (Fluorescein isothiocyanate) conjugated scrambled siRNA (C. No. sc-36869) and human ATX siRNA (C. No. sc- 44906) were from Santa Cruz Biotechnology (Santa Cruz, CA). Collection of Aqueous Humor Humans. Aqueous humor was collected from patients undergoing elective cataract surgery or surgery for glaucoma at the Duke Eye Center by the glaucoma and comprehensive clinical service clinicians. A cannula attached to a tuberculin syringe was inserted into the anterior chamber through a paracentesis tract at the start of surgery and AH was slowly aspirated until shallowing of the anterior chamber. The sample was transferred from the syringe to a microcentrifuge tube and was centrifuged at 3000 rpm for 10 min at 4uC. The supernatant was collected and stored at 280uC until further use. Animals. Mouse AH was collected from 1 year old male C57BL/6 mice. The mice were first sacrificed using sodium phenobarbital intraperitoneally and a 25 ml Hamilton syringe (22 s/20/2) was carefully inserted into the anterior chamber of the eye to withdraw the AH. Porcine eye AH was collected using a sterile insulin syringe (29G) from freshly enucleated eyes obtained from a local slaughterhouse. AH from anaesthetized (using ketamine/xylazine) New Zealand white rabbits was collected using an insulin syringe (29G). AH derived from these different species was centrifuged at 3000 rpm, 4uC for 10 minutes and the supernatant was collected and stored at 280uC till further use. Quantitative Proteomics of human AH Briefly, human AH samples were collected from 8 patients during cataract surgery, centrifuged at 3000 RPM, 4uC for 10 minutes and equal volumes of supernatant from individual specimens were pooled. Protein content of the pooled specimen was determined using a Bio-Rad dye protein assay reagent with bovine serum albumin as the standard. The AH sample was PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e42627