Ezh2 is threonine-phosphorylated. A , Ezh2 is phosphorylated in sites other than Ser-21. HEK293T cells exogenously expressing <t>FLAG-tagged</t> wild-type Ezh2 or S21A were radiolabeled with [γ- 32 P]ATP. Following immunoprecipitation using FLAG <t>antibodies,</t> bound proteins were treated with no phosphatase, with PP1, or with λ-PPase. Autoradiography ( Autorad ) confirms that both wild-type and S21A mutant Ezh2 are phosphorylated and that the signal diminishes upon phosphatase treatment. Bottom panel verifies that equal amounts FLAG-Ezh2 were used in the assay. Ctrl , control. B , Ezh2 is threonine-phosphorylated. FLAG-Ezh2 was transfected into HEK293T cells and immunoprecipitated using FLAG antibodies. Bound proteins were treated with or without λ-PPase and subjected to Western blot analysis using antibodies specific for phospho-serine (α -pSer ), phospho-threonine (α -pThr ), and phospho-tyrosine (α -pTyr ).
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