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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Metallomics. 2012 Apr 3;4(7):619–627. doi: 10.1039/c2mt20033c

Figure 6.

Figure 6

Nickel ions bind to the iron-binding site ofABH2in 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 1mMNiCl2 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. From “Nickel Ions Inhibit Histone Demethylase JMJD1A and DNA Repair Enzyme ABH2 by Replacing the Ferrous Iron in the Catalytic Centers” vol. 285(10) by Chen et al. Published, JBC Papers in Press 2009.