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Results: 4

1.
Figure 4.

Figure 4. From: Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.

Phospho-mimic Ezh2 at T345 preferentially binds to HOTAIR ncRNA in vivo. (A) Cotransfection of 293 cells with the Ezh2 mutants indicated and HOTAIR ncRNA. Transiently overexpressed Ezh2 proteins were detected by anti-Flag antibody, and CPSF-100 served as a loading control. (B) Bar graph of results from qRT–PCR of HOTAIR ncRNA bound to Ezh2 protein. Signals were normalized by the total amount of HOTAIR ncRNA in each case. (N.D.) Not detected. (C) Western analysis of stably expressed GFP-Ezh2 mutants and endogenous Ezh2 in HeLa cells using anti-Ezh2 antibody. (D) Results of RT–PCR performed on HOTAIR ncRNA bound to GFP-Ezh2 protein. PCR products were detected by qRT–PCR. (Bottom) Bar graph of qRT–PCR results for HOTAIR ncRNA with signals normalized by the total amounts of HOTAIR ncRNA in each case.

Syuzo Kaneko, et al. Genes Dev. 2010 December 1;24(23):2615-2620.
2.
Figure 2.

Figure 2. From: Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.

Ezh2 is phosphorylated by the mitotic CDK in vivo. (A) T345 and T487 of Ezh2 are phosphorylated mainly during the mitotic phase. Cells released from G1 arrest after a double-thymidine block were sampled every 2 h, and NEs were subjected to Western analyses using the antibodies indicated on the right. (B) The effect of CDK inhibitors on Ezh2 phosphorylation. HeLa cells were treated for 12 h with DMSO, 200 μM olomoucine, 20 μM roscovitine, or 50 μM etoposide. NEs were subjected to Western analyses using the antibodies indicated on the right. (C) In vitro HMKT assays of PRC2 complex containing the indicated Ezh2 mutants. Indicated amounts of PRC2 complexes were incubated with buffer containing 1 μg of recombinant nucleosomes and 1 μM 3H-SAM for 60 min. The signals were quantified by scintillation counter. (D) Western blot analysis of increasing amounts of PRC2-Ezh2 mutants using the antibodies indicated on the right.

Syuzo Kaneko, et al. Genes Dev. 2010 December 1;24(23):2615-2620.
3.
Figure 1.

Figure 1. From: Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.

CDK1 phosphorylates Ezh2 in vitro. (A) Phosphorylation sites of mouse Ezh2. Previously identified phosphorylation sites are shown above the schematic (black letters). A newly identified phosphorylation site (T345 residue) is shown below the schematic (red letter). The amino acid sequences surrounding the two phosphorylated threonine residues T345 and T487 are shown. Eed- and SUZ12-binding sites and the SET domain are indicated. (B) Schematic of the GST fusion proteins containing truncated Ezh2 ([GST-Ezh2 N] N-terminal-truncated; [GST-Ezh2 C] C-terminal-truncated) used for in vitro kinase assays. Phosphorylated threonine sites are indicated. (C, top panel) In vitro CDK1 kinase assays containing [γ-32P] ATP and the single mutant Ezh2 proteins are indicated on top. (Bottom panel) Input proteins were probed with α-GST antibody. The asterisk indicates auto-phosphorylated cyclin B. (D,E) As in B, but using cold ATP. Phosphorylated Ezh2 was detected by immunoblot using phospho-specific antibodies.

Syuzo Kaneko, et al. Genes Dev. 2010 December 1;24(23):2615-2620.
4.
Figure 3.

Figure 3. From: Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA.

Phospho-mimic Ezh2 at T345 preferentially binds to HOTAIR ncRNA in vitro. (A,B, bottom panels) Coomassie blue staining of SDS-PAGE analysis of increasing amounts of Flag tag affinity-purified Ezh2 mutants, as indicated on the right, isolated from Sf9 cells. (Top panels) Bar graphs showing the quantification by scintillation counting of P32-5′ end-labeled HOTAIR ncRNA bound to affinity-purified Ezh2. HOTAIR ncRNA was incubated with the indicated amounts of the affinity-purified Ezh2 mutants, then purified using anti-Flag M2 affinity beads. (C) Biotin-labeled HOTAIR ncRNAs were incubated with 4 μg of GST-Ezh2 N or GST-Ezh2 C proteins. Ezh2 bound to HOTAIR ncRNA was purified by Streptavidin beads and was detected by SDS-PAGE. (D) As in C, but using increasing amounts (2, 4, and 6 μg) of GST-Ezh2 1–370 or GST-Ezh2 1–341 with biotin-labeled HOTAIR ncRNA (top) or RepA RNA (bottom). The right side shows the computationally predicted structure of HOTAIR ncRNA (top) and the structure of one repeat of RepA RNA (bottom), as indicated. (E) As in C, but using increasing amounts (2, 4, and 6 μg) of full-length Ezh2, either wild type or mutant (deletion of 342–368 amino acids), as indicated. (F, left panel) Coomassie blue staining of SDS-PAGE analysis of Flag tag affinity-purified PRC2 complex isolated from Sf9 cells and containing Ezh2, either wild type or deletion mutant, as indicated. (Right panel) Western analysis of increasing amounts of affinity-purified PRC2 complex containing the indicated Ezh2 component, using the antibodies indicated on the right. (G) Domain structure of Ezh2. A newly identified domain (ncRBD1) required for ncRNA binding and containing the T345 phosphorylation site is shown in yellow. The amino acid sequence of ncRBD1 is shown below.

Syuzo Kaneko, et al. Genes Dev. 2010 December 1;24(23):2615-2620.

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