Journal: Oncotarget

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

doi: 10.18632/oncotarget.8072

Figure Lengend Snippet: Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Plasmid Preparation, Mutagenesis, Transfection

Journal: Oncotarget

Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

doi: 10.18632/oncotarget.8072

Figure Lengend Snippet: Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

Article Snippet: After cell attachment, the cells were transfected with expression vectors using FuGENE™ 6 (Promega, Fitchburg, WI), and after 48 hours of incubation, transfected 293T cells were washed with PBS and lysed by RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet-P40, 0.1 mM PMSF) with complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany).

Techniques: Incubation, Immunoprecipitation, Transfection, Western Blot