After excising the transparent cornea with scissors, the corneal epithelium was scraped off the stroma and the CE with Descemet’s membrane was peeled off as described earlier. Human CEs were plated on 100-mm dishes for culture and then were washed three times with phosphate-buffered saline (PBS), harvested, and centrifuged.
The corneal epithelium, CE, and cultured human CE were suspended in 200 μL of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), mammalian tissue protease inhibitor cocktail for 14 μM E-64 (trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane), 21 μM leupeptin, 15 μM pepstatin A, 36 μM bestatin, 0.8 μM aprotinin, and 1.4 mM AEBSF (Sigma-Aldrich, St. Louis, MO) and 1% Triton X-100, and then were sonicated for 4 minutes (Sonifier; Branson, Danbury, CT) at output level 4 and the 50% duty cycle. After centrifugation at 20,000g for 20 minutes, the protein concentration of the supernatant thus obtained was determined by a protein assay (DC; Bio-Rad Laboratories, Hercules, CA). The supernatant was diluted 1:5 in sodium dodecyl sulfate (SDS) buffer (87.5 mM Tris-HCl [pH 6.8], 600 mM DTT, 10% SDS, 0.01% bromphenol blue, and 30% glycerol) and boiled for 5 minutes. Protein extracts were also obtained from the CE of a rabbit and a C57BL/6 mouse.
In vitro translation of human and mouse CESP-1 was performed with the TNT coupled reticulocyte lysate system (Promega Co., Madison, WI), according to the manufacturer’s protocol. Because in vitro translated protein is commonly unmodified such as by phosphorylation, they were used as a positive control for immunoblot analysis. The protein was dissolved in an equal volume of SDS buffer (31 mM Tris-HCl; [pH 6.8], 4% SDS, 0.01% bromphenol blue, 20% glycerol, and 280 mM-β-mercaptoethanol and boiled for 5 minutes.