Fig. 4.

Immunoblot analysis of corneal KSPG. Corneal protein extracts were stained with Coomasie brilliant blue (CBB) (Left) and analyzed for the presence of sulfated KS-GAG by 5D4 mAb (Center) and the expression of KSPG core protein by antilumican antibody (Right). Lanes of corneal protein extracts from Chst5-WT, heterozygote, and null mice are indicated as wt, htz, and null, respectively. Each extract was incubated with (+) and without (−) endo-β-galactosidase before SDS/PAGE. A major band found in lanes of endo-β-galactosidase-digested protein on the SDS/PAGE pattern (marked by *) is exogenous BSA included with endo-β-galactosidase as a stabilizer. A prominent band (arrowhead) on the immunoblot with antilumican antibody is endogenous mouse Ig heavy chain detected by the secondary antibody. On 5D4 immunostaining, the Chst5-null lane was negative for sulfated KS GAG. Lumican protein (arrow) was detected in endo-β-galactosidase-treated lanes in corneal extracts from all three genotypes.