Journal: The Journal of Biological Chemistry
Article Title: Cyclin-dependent Kinase 1 (CDK1)-mediated Phosphorylation of Enhancer of Zeste 2 (Ezh2) Regulates Its Stability *
Figure Lengend Snippet: Ezh2 is phosphorylated at threonines 345 and 487. A , threonines 345 and 487 are conserved across species. Human, mouse, zebrafish, and fly Ezh2 were aligned using MultAlin. Relevant regions are shown. Threonines 345 and 487 are underlined , whereas conserved amino acids are highlighted in bold. B , specificity test of the phospho-Ezh2 antibodies. Dot blot analysis was performed using 2-fold dilutions of the unmodified ( Unmod. ) and phosphorylated ( Phos. ) peptides corresponding to amino acids surrounding Thr-345 or Thr-487. C , exogenously expressed Ezh2 is phosphorylated at Thr-345 ( pT345 ) and Thr-487 ( pT487 ). HEK293T cells were transfected with empty vector ( Ctrl ) or FLAG-tagged wild-type ( F-WT ), T345A ( F-T345A ), or T487A mutant Ezh2 ( F-T487A ). Following FLAG immunoprecipitation, bound proteins were analyzed by Western blot analysis using the indicated antibodies. D , endogenous Ezh2 is phosphorylated at Thr-345 and Thr-487. Western blot analysis of NIH3T3 and HeLa cell extracts using the phospho-Ezh2 antibodies was performed. For NIH3T3 extracts, 100 μg of nuclear extract ( NE ) and the equivalent volume of cytoplasmic extract ( CYT ) were loaded. For HeLa extracts, 100 μg of each fraction was loaded. Recombinant PRC2 complex was used as a positive control. Subcellular fractionation was confirmed using the following controls: tubulin (cytoplasmic), Ezh2 (nuclear), and lamin B (nuclear). NP , nuclear pellet. E , knockdown ( KD ) of Ezh2 results in decreased levels of phospho-Ezh2. HeLa cells were infected with lentiviruses expressing either control shRNA or Ezh2 shRNA. Top panel , quantitative RT-PCR confirming Ezh2 knockdown. Bottom panel , lysates were subjected to Western blot analysis using the indicated antibodies. Error bars indicate S.D.
Article Snippet: Antibodies The following antibodies were used in this study: FLAG M2 mouse monoclonal (Sigma catalog number F3165), mouse phospho-serine (Chemicon catalog number AB1603), mouse phospho-threonine (Cell Signaling catalog number 9386), mouse phospho-tyrosine 4G10 (gift from Weiguo Zhang, Duke University), rabbit Ezh2 (Cell Signaling catalog number 4905), rabbit SLBP (gift from William Marzluff, University of North Carolina), mouse cyclin B1 (Santa Cruz Biotechnology), mouse α-tubulin (Sigma catalog number T6199), lamin B (Santa Cruz Biotechnology catalog number sc-6217), mouse GST (Santa Cruz Biotechnology catalog number sc-138), rabbit H3K27me3 (Millipore catalog number 07-449), rabbit H3 (Abcam catalog number ab1791-100), and rabbit Suz12 (as described in Ref. ).
Techniques: Dot Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Western Blot, Recombinant, Positive Control, Fractionation, Infection, Expressing, shRNA, Quantitative RT-PCR