Fig. 4.

JARID1B has demethylase activity in vitro and is a Fe(II)- and α-ketoglutarate-dependent dioxygenase. (A) Histones were reacted with (lane 2) or without (lane 1) the purified ΔC fragment in the presence of all cofactors, and Western blot analysis was performed by using antibodies against tri-, di-, and monomethyl H3K4 or H3K9me3. (B) Histones were reacted with (lanes 2–6) or without (lane 1) the purified ΔC fragment in the presence of all cofactors (lane 2), with the addition of EDTA (lane 6), or in the absence of each cofactor (lanes 3–5), and Western blot analysis was performed by using an antibody against H3K4me2. Ponceau S staining of histones was used as the loading control. The arrow indicates a nonspecific protein copurified with the ΔC fragment (lower band). (C) Quantification of the data in B. (D) Histones were reacted with (lanes 2–5) or without (lane 1) the purified ΔC fragment in the presence of all cofactors and decreasing amounts of N-Oxalylglycine (N-O; 10, 1, 0.1 mM, lanes 3–5), and Western blot analysis was performed by using an antibody against H3K4me2. Ponceau S staining of histones was used as the loading control. The arrow indicates a nonspecific protein copurified with the ΔC fragment (lower band). (E) Quantification of the data in D. (F) HeLa cells transfected with the H499A mutant were immunostained with antibodies against distinctly methylated H3K4. (Left) DAPI staining. (Center) Myc staining. (Right) Methylated H3K4 staining. (Top) H3K4me3. (Middle) H3K4me2. (Bottom) H3K4me1. Arrowheads indicate cells expressing H499A mutant.