Cornea samples were immediately frozen with liquid nitrogen
after isolation from mice. To enrich the extracts with membrane-bound
proteins, TBS-CM buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM
CaCl2, and 1.5% Triton X-114) was used. Samples
were homogenized with TBS-CM buffer on ice and then centrifuged at
12,000 rpm at 4°C for 2 minutes. The supernatants were collected and
incubated at 37°C for 3 minutes, followed by centrifugation at 12,000
rpm for 2 minutes. The detergent phase, which contained enriched
membrane-bound proteins, was saved for immunoblot analysis. The
concentrations of the total protein were measured with a bicinchoninic
acid (BCA) protein assay. Equal amounts of individual samples (5 μg)
were mixed with 5 μl of 4× sample loading buffer (0.125 M Tris-HCl[
pH 6.8], 4% SDS, 40% glycerol, and 0.02% bromphenol blue)
containing β-mercaptoethanol and boiled for 5 minutes. The samples
and a prestained molecular weight marker (Bio-Rad, Cambridge, MA) were
electrophoresed on 12% SDS gels and subsequently transferred to
nitrocellulose membranes. The membranes were blocked for 30 minutes in
blocking reagent (Blotto; Santa Cruz Biotechnology, Santa Cruz, CA;
TBS, containing 0.5% Tween 20, 3% nonfat milk, and 2% bovine serum
albumin) and then incubated with a polyclonal anti-human MT4-MMP
(AB854,1 μg/ml; Chemicon, Temecula, CA) and a polyclonal anti-human
MT5-MMP antibody (AB924, 0.2 μg/ml; R&D, Minneapolis, MN),
respectively, on a rocker at room temperature for 2 hours. The species
reactivity for MT5-MMP is positive to both human and mouse according to
the information from R&D. In addition, alignment of mouse MT4- and
MT5-MMP protein sequences with that of human revealed 82.0% and 92.6%
of homology, respectively. Samples treated with nonimmune (normal)
rabbit IgG or without primary antibody treatment were processed as the
negative control. Afterward, the blot was incubated with anti-rabbit
secondary antibody conjugated with horseradish peroxidase (0.5 μg/ml;
Roche Molecular Biochemicals, Indianapolis, IN) at room temperature for
1 hour. Finally, the blot was developed by a chemiluminescence kit
(Amersham, Arlington Heights, IL), and MT4- and MT5-MMP were visualized
as dark bands with corresponding molecular weights. Immunoblot analysis
of each MT-MMP was repeated at least twice.