Expression of the ZIP1 protein in RWPE1 and RWPE2. a. Detection of the ZIP1 protein. Western blot containing 50 μg of protein extracts isolated from RWPE1 and RWPE2 cells were probed with a chicken anti-ZIP1 antibody followed by a peroxidase-conjugated secondary anti-chicken antibody. ZIP1 was visualized using an ECL kit (Amersham Biosciences). The detection of GRP78 on the same western blot was served as loading control. The protein markers (Bio-Rad) are shown. b. Quantitation of the expression level of the ZIP1 protein. The ZIP1 and GRP78 signals from the western blot (Fig. 5a) were quantified by an Alpha Innotech Gel Documentation System (Alpha Innotech). The expression of ZIP1 was normalized by the expression of GRP78. c. Regulation of the ZIP1 protein expression by zinc. RWPE1 cells were grown for 24 hours and treated with 0, 25, or 50 μM ZnSO4 for 24 hours. Cell lysate was prepared and analyzed by western blot assay. The ZIP1 and GRP78 proteins were detected as described as above.