Fig. 5
From: CoA synthase regulates mitotic fidelity via CBP-mediated acetylation
Stage-specific physical association and in vitro CBP activity assays suggest direct interaction among COASY, CBP, and TPX2. a TPX2 and b CBP showed colocalization with COASY in spindle microtubules during mitosis. COASY-GFP expressing A549 cells were stained with TPX2 (a) or CBP (b) antibody for confocal microscopy. Scale bars, 10 mm. c TPX2 and COASY physically associate with CBP during mitosis. A549 cells were synchronized by thymidine-nocodazole block and released for the indicated time to enrich cells in different cell cycle stages. An additional sample was harvested after 24 h of thymidine block for cells enriched in S phase. The CBP was immunoprecipitated, separated by electrophoresis and probed with the TPX2 and COASY antibodies. d CBP-acetylated TPX2 in vitro. Recombinant full-length TPX2 protein was acetylated by CBP (catalytic domain) human recombinant protein in the presence of acetyl-CoA. Acetylation on TPX2 was determined by western blots using pan-acetylated lysine antibody. e COASY inhibited CBP-mediated TPX2 acetylation in vitro. Bacterially purified COASY or control BSA was added to the CBP activity assay using TPX2 protein as substrate. With increasing amount of COASY, the level of acetylated TPX2 decreased in a dose-dependent manner. f Quantification of TPX2 acetylation in the presence of COASY. Acetylated TPX2 level determined by pan-acetylated lysine antibody was quantified by Image J software and normalized to TPX2 protein level. One-way ANOVA: p < 0.0001. Bonferroni post hoc tests, *p < 0.05, ***p < 0.001; n = 4 independent repeats. Bars show standard error of the mean
