Western blotting to detect MMP2 and MMP9 expression

Protein concentration of cell lysates was determined by NanoDrop ND-1000 (Thermo Fisher Scientific), and equal amounts of proteins (10 μg) of cell lysates were separated on a 10% SDS-PAGE and electrotransferred onto a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). The membranes were blocked in Tris buffered saline Tween containing 5% fat-free dry milk and then incubated with primary antibodies overnight at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 hour. Primary antibodies rabbit monoclonal to MMP-2 (1:3,000 dilution, ab192082; Abcam, Cambridge, MA, USA), rabbit monoclonal antibody to MMP-9 (1:1,000 dilution, ab194316; Abcam), and rabbit monoclonal antibody to GAPDH (1:5,000 dilution, ab181602; Abcam) were used. Proteins were detected using enhanced chemiluminescence reagents (EMD Millipore).