FIGURE 5.
FPN1 can export NTBI-derived iron. A and C, FPN1-GFP can export iron loaded as NTBI. HEp-2 cells stably expressing DMT1A-I were cultured in serum-free DMEM and preloaded with 100 μm FAC or 100 μm Fe-NTA for 6 h at 37 °C. The cells were washed with PBS, transfected with FPN1-GFP expression plasmid, and then incubated in serum-free RPMI 1640 medium for 18 h at 37 °C. The cells were lysed in TNE buffer for immunoblotting analysis (A) or in 50 mm NaOH for ferrozine-based iron assay (C). B, the density of specific bands from immunoblotting was measured with a computer-assisted imaging analysis system (NIH ImageJ), and normalization was to total protein loaded. The histogram shows the expression ratio of PCBP2, TfR1, tubulin, FPN1, GFP, and ferritin. Each column corresponds to the lane of immunoblotting analysis (A) in order from left to right. Each column is denoted as per the following labeling: (−)6 h, FAC/Fe-NTA(−) incubation 6 h; (+)6 h, FAC/Fe-NTA(+) incubation 6 h; (+)18 h, FAC/Fe-NTA(+) incubation 6 and 18 h; and (+)18 h FPN/GFP, FAC/Fe-NTA(+) incubation 6 h and FPN1 or GFP transfection 18 h. Significance was determined using Student's t test: †, p < 0.05; and §, p < 0.001. C, the histogram shows the relative intracellular iron level. The detailed method is described under “Experimental Procedures.” The results in the histogram are presented as the means ± S.E. of three independent experiments. Significance was determined using Student's t test: †, p < 0.05; §, p < 0.001.