Enhanced chemiluminescence detection kit

For lysate preparation, tissues or cultured cells were washed twice with PBS and then placed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Sigma, St. Louis, MO). The lysate protein concentration was determined using a bicinchoninic acid assay kit. Western blot analysis was performed using standard techniques. Equal amounts of lysate protein (30 μg) in the RIPA buffer were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and the proteins were electrophoretically transferred to nitrocellulose membranes. The membranes were blocked in 5% non-fat dried milk in TBS containing 0.1% Tween-20 (TBS-T) for 1 h, and then incubated overnight at 4°C with primary polyclonal antibodies raised against MMP-3, MMP-13, or β-actin; these were all diluted in TBS-T containing 5% dried milk. The primary antibodies were detected by incubation with horseradish peroxidase-conjugated secondary antibodies for a further 1 h. Specific antibody binding was visualized using an enhanced chemiluminescence detection kit (ELPIS Biotech, Korea) and X-ray film exposure. The presented data were representative of at least three independent experiments. Densitometric analyses were conducted using Image J software.