Extended Data Fig. 3: HDAC3 interacts with and decrotonylates EB1.
From: Dynamic crotonylation of EB1 by TIP60 ensures accurate spindle positioning in mitosis
(a) FLAG-HDAC1-3 or FLAG-SIRT1-3 were co-transfected with EB1-GFP in HEK293T cells, and their interactions were tested by co-immunoprecipitation. (b) Whole-cell Lysates (WCL) of HeLa cells expressing FLAG-HDAC3 wild type or deacetylase-dead point mutants (Y298H and VRPP) were probed with indicated antibodies, including site-specific modification of antibodies. (c) Whole-cell Lysates (WCL) of HeLa cells expressing HDAC3 shRNA or FLAG-HDAC3 were probed with an anti-CrK66 antibody to assess the crotonylation level of EB1. (d) FLAG-TIP60 was incubated with His-EB1 in the presence of indicated concentrations of Cr-CoA or Ac-CoA for in vitro acylation assay. The acetylation and crotonylation levels of EB1 were analyzed with anti-AcK66-EB1 and CrK66-EB1 antibodies. Purified proteins were validated via CBB staining. Related to Fig. 1.
