X ray film

Cells and tissues were lysed in triton lysis buffer (25 mM sodium phosphate, 150 mM sodium chloride, 1% Triton X-100, 5 mM EDTA, 50 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 5 μM pepstatin A, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 25 μM phenylarsine oxide) for 30 min at 4 °C. Aliquots of 50 micrograms in Laemmli buffer were heated in boiling water for 5 min, electrophoresed on 10% polyacrylamide gels, and transferred to PVDF membranes (Biorad, Hercules, CA). The membranes were treated for one hour at room temperature with blocking buffer (5% milk proteins, 0.05% Tween 20 in Tris buffer, pH 8.1) and hybridized overnight in the same buffer containing 1:500 dilutions of rabbit anti-Neu (SC-284) and rabbit anti-GAPDH (SC-25,778) (both antibodies from Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were washed 3 times for 5 min with 0.05% Tween-20 in Tris-Cl, pH 8.1 and probed with a 1:2000 dilution of goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, SC-2004) in blocking buffer for 1 h at room temperature. The membranes were then washed 3 times for 10 min in 0.05% Tween 20 in Tris buffer, pH 8.1 and incubated with ECL Western blot Substrate (ThermoFisher, Grand Island, NY, Catalog Number 32106) for 1 min before being exposed to X-ray films (Denville Scientific, Holliston, MA, Catalog Number E3012) for 5–10 min and developed.