Fig 1: AhR, PKM2, PDC-E2 and p300 constitute a complex in the chromatin fraction. (A) eAhR complex was purified from a solubilized chromatin fraction of stably transduced HeLa S3 cells after treatment with 100 nM indirubin for 3 h, and analyzed by western blotting. A mock purification was performed using non-transduced HeLa S3 cells. (B) ePKM2 complex was purified from a solubilized chromatin fraction of stably transduced HeLa S3 cells after treatment with 100 nM indirubin for 3 h and analyzed by western blotting. A mock purification was performed using non-transduced HeLa S3 cells. (C) Endogenous AhR was immunoprecipitated by anti-AhR antibody from the chromatin fraction of HeLa S3 cells. (D) Endogenous PDC-E2 was immunoprecipitated by anti-PDC-E2 antibody from the chromatin fraction of HeLa S3 cells. (E) eAhR complex was immunoaffinity-purified by anti-FLAG antibody-conjugated agarose from whole-cell lysates of eAhR-expressing HeLa S3 cells not treated (-) or treated (+) with 100 nM indirubin for 3 h. F. PKM2 complex was immunoprecipitated using anti-PKM2 antibody from whole-cell lysates of HeLa S3 cells not treated (-) or treated (+) with 100 nM indirubin for 1 h.
Fig 2: The nucleus has PDC activity and PDC-E2 is recruited to the CYP1A1 enhancer in an AhR-ligand-dependent manner. (A) HeLa S3 cells were fractionated into cytoplasmic, mitochondrial and nuclear fractions. The nuclear fraction was further purified by sucrose density-gradient centrifugation as described in the Experimental procedures section. Lysates of each fraction were subjected to western blotting. (B) Kinetics parameters of acetyl-CoA production from pyruvate in each fraction. Each fraction was subjected to a PDC assay as described in the ‘Materials and Methods’ section, and the apparent Km for pyruvate and apparent Vmax were determined (mean ± SE, n = 3). (C) HeLa S3 cells were exposed to 100 nM indirubin for 45 min. ChIP assays were performed with IgG or anti-PDC-E2 antibody (mean ± SD, n = 3). *P < 0.05, ***P < 0.001 (Student's t-test).
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