Fig. 3
From: Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients
GCN5 acetylates and SIRT3 deacetylates ECHS1. a Myc-tagged GCN5 and Flag-tagged ECHS1 were co-expressed in HEK293T cells. The co-purified Myc-tagged GCN5 was detected in Flag beads purified proteins. b, c Western blot detection of endogenous ECHS1, and AcK and AcK101 levels of affinity-purified ECHS1 from HEK293T cells and GCN5 overexpressing HEK293T cells (b) and from HEK293T cells and GCN5 knockdown HEK293T cells (c). d Western blot detection of AcK and AcK101 levels of recombinant ECHS1 that before and after in vitro acetylation by GCN5. e The levels of endogenous ECHS1 were detected in HEK293T and GCN5 knockdown HEK293T cells that were cultured in the absence of glucose or presence of glucose. f The synthetic acetylated K101-containing ECHS1 peptide was deacetylated by recombinant SIRT3. The synthetic peptide (1047.5) and de-acetylated products (1005.4, red-circled) were assayed by MS. g The levels of AcK and AcK101 of hyperacetylated ECHS1 (from NAM treated cells) were detected before and after in vitro treatment by SIRT3. h ECHS1 was co-expressed with SIRT3 or SIRT3H248Y in HEK293T cells. The AcK101 levels of purified ECHS1 were determined. i The Ac101 levels of affinity-purified ECHS1 from liver of Sirt3 KO and control mice
