Western blot (immunoblot) analysis

Whole-cell protein extracts were subjected to a 10% SDS-PAGE and transferred to an ECL-Hybond membrane (GE Healthcare Life Sciences, Little Chalfont, UK) using a mini-electroblot (Biorad, Hercules, CA, USA). After blocking for 2 h in 5% nonfat dry milk in TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Tween 20) the membranes were incubated overnight with mouse polyclonal anti-actin (1:1500 dilution, Santa Cruz, Dallas, TX, USA), anti-GAPDH (1:500 dilution Santa Cruz), mouse monoclonal anti-p-p38 (1:200 dilution, Santa Cruz), anti MMP2 (1:1000 dilution, Genetex, Irvine, CA, USA). Peroxidase conjugated anti-rabbit, anti-mouse and anti-rat (1:5000 dilution; Promega, Fitchburg, WI, USA) were used as secondary antibodies. Images are acquired with the Versa-Doc Imaging System (Bio-Rad) and analyzed with the Quantity One software (Biorad).