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completely dried before tryptic digestion in sequencing grade trypsin solution (12.5 μg/μL; Thermo Fisher Scientific Inc, Rockford, IL, USA) in 50mM ammonium bicarbonate. The digestion was performed at 37 °C overnight, and then the extraction step was performed once with 25mM ammonium bicarbonate, twice with 5% formic acid, and then once with distilled water. The extracted peptides were pooled and dried. After re-sus-pending in 40 μL of aqueous 0.01% trifluoroacetic acid/2% acetonitrile, the samples were analyzed by liquid chromatography-mass spectrometry (LCQ Deca XP plus, Thermo Fisher Scientific Inc).Proteomic analysis of monkey CBDatabase searches were performed with the assistance of Bio Works 3.3.1, a protein search program. The UniProt-SwissProt database was initially used by querying the entire theoretical peptide masses provided in the public domain by the Swiss Institute of Bioinformatics. The number of registrations in the UniProt-SwissProt database is about 17 000 for humans and about 14 000 for mice. The data analyses of the monkey CB were aimed at cynomolgus monkeys, humans, orangutans, chimpanzees, rhesus macaques, and lowland gorillas.Immunohistochemistry of the CBEnucleated eyes from normal cynomolgus monkeys were fixed in 10% neutralized and buffered formal-dehyde solution at 4 °C overnight and then dehy-drated. The eyes were embedded in paraffin and serially sectioned at 4 μm thickness. After deparaf-finization and rehydration, the specimens were prepared for antigen retrieval by warming in hot water in Target Retrieval Solution (Dako, Glostrup, Denmark) for 30 minutes at 100 °C. The sections were then blocked with phosphate-buffered saline (PBS) containing 10% BSA for one hour and then incubated overnight with primary antibodies (Abs) that were the same as those used for the Western blotting. The slides were washed in PBS and, for nuclear staining, were incubated with Alexa 488 or Alexa 568 (1:500 dilution; Invitrogen) and 4’,6’- diamidino-2-phenylindole (DAPI) for one hour at room temperature. The stained tissues were exam-ined with a confocal fluorescence laser microscope (Radiance 2000, Bio-Rad Laboratories). Control slides were prepared by a similar process, but the primary Abs were omitted.Primary culture of ciliary epithelial cells from cynomolgus monkeyThe ciliary epithelial cells isolated from cynomo-lgus monkey eyes were suspended in DMEM (GIBCO, Carlsbad, CA, USA) containing penicil-lin-streptomycin (100 U/mL final concentration; Invitrogen) and 10% FBS. Cells were grown to confluence at 37 °C in 5% CO2. Then, cells were cultured with 100 nM of the glucocorticoid dexa-methasone (DEX), 500 nM of timolol malate, or 1 μM of acetazolamide (Sigma-Aldrich, St Louis, MO, USA) for five days. Protein expression of lysate from blotting of CBCultured monkey ciliary epithelial cells were lysed in TNE buffer containing 50mM Tris, 137mM NaCL, 1mM EDTA, 1% TritonX-100, and protease inhibitors (Complete EDTA-free; Roche, Basel, Switzerland). After homogenization and centrifu-gation for 15 minutes at 14 000 rpm, the superna-tant was collected. The protein concentration was determined with the RC-DC protein assay kit (Bio-Rad) according to the manufacturer’s instruc-tions. Ten micrograms of proteins from the monkey ciliary epithelium cells lysates were diluted in an equal volume of 2×Laemmli buffer and heated for 5 minutes at 100 °C. Samples were separated by 7.5% SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membrane. Membranes were blocked in PBS containing 0.05% tween20 (PBS-T) and 5% non-fat dry milk for one hour and probed overnight at 4 °C with one of the following primary Abs: rabbit anti-Rab8 Ab (Sigma-Al-drich), goat anti-Ezrin Ab (Sigma-Aldrich), goat anti-Radixin Ab (Sigma-Aldrich), goat anti-Moesin Ab (Sigma), and mouse anti-Actin Ab (Millipore, Billerica, MA, USA). The specific signals were detected with horseradish peroxidase (HRP)- conjugated donkey Ab to goat IgG or rabbit IgG as secondary Abs. The signals were made visible by chemiluminescence reactions and examined with a ChemiDoc XRS plus (Bio-Rad).Proteomic analysis of monkey CBThe proteomic analysis of proteins extracted from the stroma and epithelium of the monkey CB 341Results