Fig. 4
From: CoA synthase regulates mitotic fidelity via CBP-mediated acetylation
CBP acetylates and stabilizes TPX2 protein level. a COASY knockdown increased two CBP-acetylated peptides that cover K1762. b CBP-acetylated TPX2 in vivo. Wild type or catalytic-deficient mutant of HA-tagged CBP was cotransfected with V5-tagged TPX2 into HEK-293T cells. After 20 min of release from thymidine-nocodazole block, the V5-tagged TPX2 was then immunoprecipitated with control (IgG) or V5 antibody (V5) and then probed with HA (CBP), acetylated lysine or V5 (TPX2). Wild type, but not catalytic-deficient mutant, CBP increased TPX2 acetylation and levels. c CBP increased the half-life of TPX2. Wild type or catalytic-deficient mutant of CBP was cotransfected with V5-tagged TPX2 into unsynchronized HEK-293T cells. After 24 h, the protein synthesis of the transfected cells was halted with 25 mg/ml cycloheximide and collected at the indicated times for western blots. The TPX2 protein level at indicated time points was quantified by Image J software and normalized to b-tubulin protein level. d CBP siRNA abolished the increased TPX2 and Aurora-A phosphorylation caused by COASY knockdown. A549 cells transfected with control, siCOASY or siCBP were released from thymidine-nocodazole block for 20 min and probed for western blots using indicated antibodies. The phosphorylated Thr 288 of Aurora A indicates activation. e CBP knockdown rescued the multinucleation caused by COASY knockdown. Multinucleation was determined by immunofluorescence. f CBP knockdown decreased TPX2 protein level during mitosis. A549 cells transfected with siRNA targeting CBP were synchronized by thymidine-nocodazole block and released in fresh media. The released cells were harvested every 20 min and probed with indicated antibodies. To examine TPX2 protein level in S phase, additional samples were harvested after 24 h of thymidine block. Bars show standard error of the mean. a, e N.S. not significant ***p < 0.001, **p < 0.01, two-tailed Student’s t-test, n = 3 independent repeats. c Two-way ANOVA: p < 0.0001. Bonferroni post hoc tests, ***p < 0.001, n = 4 independent repeats
