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in aqueous humor production. Besides aqueous humor formation, the CB is involved in accommodation and anterior cham-ber-associated immune deviation. The CB, espe-cially the non-pigmented ciliary epithelium, is also involved in the synthesis and secretion of various proteins found in the aqueous humor that are believed to be involved in controlling IOP4-6). Proteins secreted from the CB can affect the trabecular meshwork cells. The glycoproteins in aqueous humor have been shown to be secreted from the CB epithelium7), and molecules in the aqueous humor, e.g., collagenases, can affect the trabecular meshwork morphology8, 9). However, a comprehensive description of the proteins present in the aqueous humor has not been published. Recent advances in proteomics technology have made it possible to perform comprehensive investi-gations of the proteins in body fluids and tissues. In ophthalmology, proteomics studies have analyzed the proteins in the cells of the trabecular mesh-work, aqueous humor, retina, retinal pigment epithelium, and retinal drusen10-17).To date, no study has comprehensively deter-mined the proteins expressed in the CB. In addi-tion, the many steps involved in the synthesis and secretion of aqueous humor have not been deter-mined. Therefore, the purpose of this study was to determine the proteins present in the CB. To accomplish this, we conducted a proteomic analysis of the CB of cynomolgus monkeys and thereby focused on the small guanosine triphosphate (GTP)-binding protein Rab8, which has been shown to be involved in vesicle transport and protein localization in small intestinal epithelial cells. Rab8 is also involved in the morphology of the microvilli in small intestinal epithelial18) and is asso-ciated with optineurin19, 20), a glaucoma gene. Materials and methodsPreparation of cynomolgus monkey eyesAll experimental procedures were approved by the Animal Welfare and Animal Care Committee of the Tsukuba Primate Research Center (TRPC) and the Experimental Animal Committee of the National Tokyo Medical Center. The facilities for housing the monkeys are accredited by the Associ-ation for Assessment and Accreditation of Labora-tory Animal Care International (AAALAC Inter-340national). Monkeys are routinely examined for physical and ophthalmic conditions by veterinar-ians and ophthalmologists, and all experiments on monkeys are conducted in accordance with The Association for Research in Vision and Ophthal-mology Statement for the Use of Animals in Ophthalmic and Vision Research.Eyes were obtained in a collaborative study to make effective use of all the monkey tissues for different research programs. Eyes were enucleated immediately after the monkeys were euthanized, and the CBs were isolated from the eyes within 4 hours after death. Protein extraction from CBsThe tissues from the CBs of a healthy male monkey were homogenized and sonified in lysis buffer (50 mM Tris-HCL, 2 mM EDTA, 0.5% Triton-X, 2% SDS). After centrifugation for 10 minutes at 10 000 rpm (9,300g), the supernatant was collected. The protein concentration in the supernatant was determined with the RC DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer’s instruc-tions. Twenty micrograms of the protein sample were combined with an equal volume of 2 × Laemmli buffer and heated for 5 minutes at 100 °C. Then, all samples were stored at -20 °C until use.Gel digestion and liquid chromatography-mass spectrometry analysesTwenty micrograms of protein sample was sepa-rated on 12.5% acrylamide SDS-PAGE gel. The gel was stained with Colloidal Coomassie Blue (Invit-rogen, Carlsbad, CA, USA) and cut into 15 equal pieces of approximately 1 mm3. The pieces were washed twice with 50mM ammonium bicar-bonate/50% acetonitrile, and after destaining, the gel pieces were rinsed with distilled water and incubated with acetonitrile for 20 minutes. The supernatant was discarded, and the gel pieces were completely dried before incubation with 10mM DTT in 100mM ammonium bicarbonate for 45 minutes at 56 °C. The supernatant was discarded, and the pieces were incubated in the dark with 55mM iodoacetamide in 100mM ammonium bicar-bonate for 30 minutes at room temperature. Then, the supernatant was discarded, and the gels were washed three times. Finally, the gel pieces were