Figure 1.

SET7/9 methylates JMJD2B. A. Immunoprecipitation (IP) of Flag-tagged JMJD2B with anti-Flag antibodies from 293T cells upon coexpression with wild-type SET7/9 or its catalytically inactive H297A mutant. Methylation was assessed with p53-K372me antibodies (top), while the bottom three panels show Western blots with the input levels of Flag-JMJD2B, SET7/9 or actin. B. Immunoprecipitation of endogenous JMJD2B from 293T cells; IgG and anti-RCL antibodies served as negative controls. C. In vitro methylation of JMJD2B by GST-SET7/9 utilizing radioactive ³H-SAM. Either GST or fusions of GST to indicated JMJD2B amino acids (asterisks mark the respective full-length proteins, but smaller polypeptides that were likely due to proteolytic degradation are also visible in the Ponceau stains) were used as substrates. The top shows a sketch of JMJD2B with its four known domains: catalytic jmjC, accessory jmjN, double PHD and double Tudor domains.