Whole-cell extracts were prepared from both rat corneal epithelium
and cultured THCE cells by lysing tissues and cells in RIPA buffer (150
mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl
sulfate [SDS], 50 mM Tris, [pH 8.0]) with protease inhibitors
leupeptin, aprotinin, pepstatin A, soybean trypsin inhibitor (5 μg/ml
each), and 1 mM phenylmethylsulfonyl fluoride. The protein
concentrations were determined by a protein assay reagent kit (Micro
BCA; Pierce, Rockford, IL). Equal amounts of protein were mixed with
SDS-sample buffer and boiled for 5 minutes before loading. Proteins
were separated by 5% to 15% gradient SDS-PAGE and transferred
electronically to the nitrocellulose membranes. The quality of the
transfer was monitored by ponceau S staining. After blocking with 5%
nonfat milk, the membranes were incubated with polyclonal antibodies
against occludin, ZO-1, ZO-2, and claudin-1 (Zymed, 1:1000 dilution in
5% nonfat milk) in TBST. Horseradish peroxidase–conjugated goat
anti-rabbit IgG (Bio-Rad, Hercules, CA) was applied for 1 hour. Immune
complexes were visualized with an enhanced chemiluminescence reagent
(Pierce). Results were quantified by capturing the exposed x-ray film
image with BDS Image System (Biological Detection System, Pittsburgh,
PA), and using area measurements from image analysis software
(NIH Image, ver. 1.60; National Institutes of Health, Bethesda, MD).
Experimental values were within the linear range of the assay.