“…Membranes were blocked with Tris-buffered saline with Tween-20 (TBST) (0.1%Tween-20) plus 5% non-fat dry milk overnight at 4 C with gentle shaking, washed with TBST three times (15 min each), and probed with primary antibodies overnight at 4 C [rabbit anti C-terminus of human HB-EGF, 1:200; goat anti N-terminus of human HB-EGF, 1:200, R&D Systems (Minneapolis, MN); sheep anti-ADAM 12, 0.1mg/ml, R&D Systems], followed by incubation with HRP-conjugated secondary antibodies 1 h at room temperature (goat anti-rabbit IgG; rabbit anti-goat IgG, or rabbit anti-sheep IgG, Jackson ImmunoResearch Laboratories, West Grove, PA). Specific immunoreactivity was determined by application of SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL) to the membrane and normalized to actin (Provenzano et al, 2005).…”