Protein concentrations of ShODC or DFMO-treated ESCC cell lysates were determined using a protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA). Total proteins (20 to 100 μg) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). After blocking in 5% non-fat milk, the membranes were probed with specific primary antibodies overnight at 4 °C, then washed 3 times with TBS-Tween 20 followed by incubation at room temperature 1 h with a horseradish peroxidase (HRP)-conjugated secondary antibody. The protein bands were visualized with a chemiluminescence reagent (GE Healthcare Biosciences, Pittsburgh, PA).