Western blot analysis investigating the level of total ERK 1/2 and pERK 1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in CECs were performed as follows. CECs were cultured as previously and then exposed to either a 150-mV/mm EF or 100 ng/mL HGF for 15, 30, and 60 minutes. Cells were harvested using a lysis buffer (2% SDS, 70 mM Tris-HCl [pH 6.8]), aspirated, and boiled for 20 minutes. Cell extracts were normalized for total protein with a protein assay (Bio-Rad Laboratories, Hemel Hempstead, UK) to enable 10 μg of each sample to be electrophoresed through a 4% to 20% polyacrylamide gradient gel (Bio-Rad). The sample proteins were transferred to nitrocellulose membrane (ECL; Amersham International, PLC, Little Chalfont, UK) and the membranes blocked for 2 hours with 5% nonfat milk in TBS-T (TBS with 0.1% Tween-20). The membranes were incubated with mouse monoclonal anti-pERK 1/2 or rabbit polyclonal total ERK 1/2 (Santa Cruz Biotechnology, Inc.) at a 1:1000 dilution for 1 hour and then biotinylated anti-mouse secondary or anti-rabbit secondary antibodies (SAPU, Carluke, Scotland, UK) at a 1:10,000 dilution for 1 hour. Streptavidin conjugated to horseradish peroxidase (SAPU) was added last at a 1:10,000 dilution. The blots were developed using o-dianisidine stain, and the dried membranes were scanned into a computer (Photoshop 5.02; Adobe, San Jose, CA) to prevent photobleaching.