Acetylation promotes TcHMGB re-localization from the nucleus to the cytoplasm and shedding to the extracellular medium.

(A) Immunofluorescence assay using purified anti-TcHMGB antibody and parasites at different stages of the T. cruzi life cycle: Epimastigotes, Free Amastigotes released from infected Vero cells and Trypomastigotes treated (+) and non-treated (-) with deacetylase inhibitors (iDAC) Trichostatin A, Sodium Butyrate and Nicotinamide. Anti-rabbit IgG conjugated to fluorescein was used as a secondary antibody. Nuclei and kinetoplasts were labeled with DAPI. Bars = 2 μm. (B) Slot western blot assay. Culture supernatants in FCS-free media were treated (+) and non-treated (-) with deacetylase inhibitors (iDAC) and blotted onto nitrocellulose membranes. Epimastigotes: E, Infected Vero cells: I C, Trypomastigotes: T, and un-infected Vero cells: UI C. Rabbit anti-TcHMGB (aTcHMGB) antibodies were used to detect the presence of TcHMGB. Recombinant TcHMGB (rTcHMGB) was used as positive control.