Lens epithelial cells were lysed on ice in lysis buffer (20 mM
Tris-HCl [pH 7.5], 120 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM
EDTA, 2 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 20 μM leupeptin, and 1 μM aprotinin; Sigma). For sodium
dodecyl sulfate (SDS)- and urea-soluble fractions, cells were lysed in
buffer containing 1% SDS and 8 M urea, respectively. After
centrifugation for 10 minutes at 10,000g, the supernatant
was stored at −20°C. Protein concentrations were determined by using
a protein assay kit (DC; Bio-Rad Laboratories, Hercules, CA). The
lysates containing 20 μg protein were boiled for 5 minutes in SDS
sample buffer, fractionated by SDS−10% polyacrylamide gel
electrophoresis, and transferred to a nitrocellulose membrane (Hybond;
Amersham Life Science, Cleveland, OH) using an electroblot apparatus
(Bio-Rad). Membranes were blocked for 1 hour in
Tween–phosphate-buffered saline (PBS-Tween) containing 5% nonfat milk
powder. The membranes were then incubated with primary antibody at
1:1000 dilution in PBS-Tween containing 5% nonfat milk powder at room
temperature for 45 minutes and washed in PBS-Tween. The primary
antibodies used in this study were rabbit anti-human fibronectin
antibody (Sigma) and mouse anti-human smooth muscle actin (SMA)
antibody (Sigma). Blots were then incubated with a 1:1000 dilution of
horseradish peroxidase–conjugated anti-rabbit or anti-mouse antibody
(Amersham) at room temperature for 45 minutes After they were washed
three times in PBS-Tween, the blots were developed by using chromogenic
substrate solution (diaminobenzidine substrate; Boehringer Mannheim).
Prestained molecular weight standards (SeeBlue) were purchased from
Novex.