To find differences in the expression of transforming growth factor-beta-induced protein (TGFBIp), a major component of GCD2 corneal deposits, western blot analysis of primary cultured corneal fibroblasts was performed on ten different donated normal corneas. Corneas were collected from a 37 year-old man (normal corneal fibroblasts (NCF-1), a 62 year-old man (NCF-2), a 20 year-old woman (NCF-3), a 46 year-old man (NCF-4), a 29 year-old man (NCF-5), a 60 year-old man (NCF-6), a man with unknown-age (NCF-7), a woman with unknown-age (NCF-8), a 69 year-old woman (NCF-9), and an 81 year-old man (NCF-10). We first performed western blot analysis of four corneas (Figure 1A) to find out the variable expression level of TGFBIp from NCF-1, 2, 3, and 4 and the expression level was very low in NCF-3. We performed western blot analysis of another six corneas from normal donors (Figure 1B). Western blot analysis of NCF-4 was repeated in the second experiment to allow comparison between the two separate experiments.
To measure the level of TGFBIp expression, total cellular protein was electrophoresed on Tris-glycine SDS polyacrylamide gels. Equal amounts of proteins were then transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA), blocked in 5% dry milk in Tris-buffered saline containing Tween-20 (TBS-T) at room temperature for one hour, and then incubated with primary antibodies to TGFBIp (0.2 microg/ml; Cat. No. AF2935; R&D Systems, Minneapolis, MN) and beta-actin (1:5,000 dilution; Cat. No. A-5441; Sigma, St Louis, MO) overnight at 4  C. After three washes with TBS-T, blots were incubated with secondary antibodies conjugated to horseradish peroxidase at room temperature for 1 h. Horseradish peroxidase-linked anti-mouse IgG (1:5,000 dilution; Cat. No. 31460) or anti-goat IgG (1:5,000 dilution; Cat. No. 31430) were used as a secondary antibody (Pierce, Rockford, IL). Western blots were visualized using the enhanced chemiluminescence system (Pierce).
This study was approved by the Severance Hospital Institutional Review Board and informed consent was obtained from all subjects participating in this study.