Whole-cell lysates or concentrated medium in 2× sample buffer were electrophoresed into 14% or 10% polyacrylamide gels containing 0.1% SDS. Fractionated proteins were transferred to nitrocellulose by a commercial system (Mini Transblot; Bio-Rad, Hercules, CA). Blots were blocked for 1 hour at approximately 25°C in Tris-buffered saline (137 mM NaCl, 25 mM Tris, and 2.7 mM KCl) containing 0.2% Tween-20 (TBST) and 5% (wt/vol) nonfat dry milk and then probed with anti-MMP-2 IgG (1:1000), anti-MMP-3 IgG (1:1000), anti-MMP-9 IgG (1:200), anti-MMP-1 IgG (1:200), anti-TIMP-1 IgG (1:1000), anti-TIMP-2 IgG (1:200), anti-ERK1/2 IgG (1:1000), or anti-phosphoERK1/2 (1:1000) overnight at 4°C (MMP/TIMP IgG from Oncogene Research Products, Boston, MA; ERK antibodies from Cell Signaling Technologies, Beverly, MA). The blots were washed (four times for 15 minutes each) in TBST and incubated for 1 hour at 25°C with horseradish peroxidase–conjugated secondary antibodies in 5% milk in TBST (goat anti-mouse, 1:5000; goat anti-rabbit, 1:5000). The blots were washed in TBST (four times for 15 minutes each), and enhanced chemiluminescence (Amersham Biosciences, Arlington Heights, IL) was used to visualize specific labeling. Blots were digitized with a gel-documentation system (EpiChemi II Darkroom; UVP, Upland, CA), and densitometry was performed on computer (Laboratory Works imaging software, ver. 4.0.0.8; Ultraviolet Products Inc., Upland, CA).