Article Title: KAT2A coupled with the α-KGDH complex acts as a histone H3 succinyltransferase
Figure Lengend Snippet: KAT2A interacts with the α-KGDH complex in the nucleus a , OGDH binds to KAT2A. KAT2A was immunoprecipitated from U251 cells using an anti-KAT2A antibody. The immunoprecipitate was analysed by mass spectrometry. A specific peptide (920-DMVGQVAIT-929) of OGDH was identified. The IonScore is 30, the q value is 0.004, and the Exp value ( P ) is 0.1792. One-sided t -tests were conducted. Representative results of two experiments are shown. b , Quantitative analyses of α-KGDH subunits in the nucleus. Immunoblotting analyses of α-KGDH subunits in the nucleus extraction of U251 cells were performed with the indicated antibodies displayed in . The quantitative standard curves were used to determine the percentage of nuclear OGDH, DLST, and DLD. The x -axis presents the intensities of quantified proteins detected by immunoblotting analysis, and the y -axis presents the percentage of whole-cell lysate corresponding to each quantified protein. The percentages of nuclear OGDH, DLST, and DLD were calculated using the equations displayed above the standard curves. Representative images of triplicate experiments are shown. The R 2 values represent the relationship between log Y and X to the linear model (log Y = log[ A + B ] × X ). Y , percentage of whole cell lysate; X , intensity. c , Nuclear distribution of the α-KGDH complex. Immunofluorescent staining of U251 cells was performed with anti-OGDH, anti-DLST, or anti-DLD antibodies (green). The mitochondria and nuclei were stained with an anti-Tom20 antibody (red) and DAPI (blue), respectively. Representative images of more than 50 cells are shown. The majority of the α-KGDH complex overlapped with the mitochondrial protein Tom20, indicating primary mitochondrial localization of the α-KGDH complex. d , Quantitative analyses of the interaction between KAT2A and α-KGDH subunits. Immunoprecipitation analyses of U251 cell nuclei with anti-KAT2A or anti-OGDH antibodies were performed. KAT2A-or OGDH-associated proteins were analysed via immunoblotting analyses. Representative images of triplicate experiments are shown. e , f , KAT2A is not localized in mitochondria. e , Immunoblotting analyses of the mitochondria extraction and total cell lysate of U251 cells were performed with the indicated antibodies. Representative images of triplicate experiments are shown. f , Immunofluorescent analyses of U251 cells were performed with DAPI (blue) and anti-KAT2A (green) and anti- TOM20 (red) antibodies. Representative images of more than 50 cells are shown. g , OGDH depletion, which does not affect the interaction between DLST and DLD, inhibits the binding of DLST to KAT2A. OGDH was depleted in U251 cells. Immunoprecipitation analyses with an anti-DLST antibody were followed by immunoblotting analyses with the indicated antibodies. Representative images of triplicate experiments are shown. h , i , The NLS of DLST is required for nuclear distribution of the α-KGDH complex. DLST was depleted from U87 cells, which were reconstituted with expression of wild-type rDLST or the rDLST(R224A/K226E) NLS mutant. h , Immunofluorescent staining was performed with an anti- DLST or anti-OGDH antibody. The mitochondria and nuclei were stained with an anti-Tom20 antibody (red) and DAPI (blue), respectively. i , Immunoblotting analyses were performed with the indicated antibodies. Mitochondrial COX IV and IDH2 and nuclear PCNA were used to show lack of mitochondrial contamination of nuclear fractions and loading controls. Representative images of triplicate experiments are shown. Fig. 1b
Article Snippet: Parental U87, U251, and 293 cells were obtained from the American Type Culture Collection.
Techniques: Immunoprecipitation, Mass Spectrometry, Staining, Binding Assay, Expressing, Mutagenesis