Goat anti rabbit or anti mouse igg horseradish peroxidase

Cells were lysed in Laemmli buffer and stored at À70 °C. Protein concentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Samples (20-30 lg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Hybond â P; GE Healthcare, Little Chalfont, UK). Membranes were blocked for 1 h at room temperature with TBST (20 mM Tris, 137 mM NaCl, 3.8 mM HCl, and 0.1% [v/v] Tween â 20) containing 5% (w/v) nonfat milk powder, and blots were probed with anti-ID1, anti-ID2 or anti-ID3 (Z-8, C-20, C-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-cadherin (HECD-1; Abcam, Cambridge, UK), anti-N-cadherin (32; BD Biosciences, Franklin Lakes, NJ, USA), anti-vimentin (V9; Dako, Glostrup, Denmark), anti-Snail (ab117866;
Abcam, Cambridge, UK), anti-MMP-9 (ab35326; Abcam) or anti-actin (C4; EMD Millipore, Billerica, MA, USA) antibodies for 1 h. Membranes were washed and incubated with a secondary antibody (either goat antirabbit or anti-mouse IgG-horseradish peroxidase) (Santa Cruz Biotechnology), washed again and developed for enhanced chemiluminescence using the Amersham ECL-Plus kit according to the manufacturer's instructions (GE Healthcare).