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. 2009 Dec 30;285(10):7374–7383. doi: 10.1074/jbc.M109.058503

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FIGURE 7. — Nickel ions bind to the iron-binding site of ABH2 in cells. A, measurement of FLAG-ABH2 and ABH2(D173A) expression levels in the nickel-treated 293T cells. 293T cells were transiently transfected with FLAG-ABH2 and FLAG-ABH2(D173A) expression vectors and then treated with 1 mm NiCl₂ that contained 0.22 mCi of ⁶³NiCl₂. Expression of FLAG-ABH2 or ABH2(D173A) in cell lysates was measured by Western blot using anti-FLAG antibody. The intensity of bands was quantified using ImageJ software and marked below the graph. The quantification results were graphed on the right. B, cell lysates collected in A were subject to IP with anti-FLAG resin. The FLAG-tagged recombinant proteins were eluted with FLAG peptide, and their associated radioactivity was measured. C, ⁶³Ni-specific radioactivity associated with FLAG-ABH2 or ABH2(D173A) was calculated. The experiment was conducted in triplicate, and values are means ± S.D. for triplicates. The difference in ⁶³Ni-specific radioactivity between FLAG-ABH2 and FLAG-ABH2(D173A) samples is statistically significant because a two-tailed Student t test analysis gives a p value of 0.044.