Detyrosinated α tubulin

For immunohistochemistry assay, the heart tissue was frozen in OCT in liquid nitrogen and sectioned at 0.5 μm. The sections were incubated with a primary antibody against detyrosinated α-tubulin (Abcam, Cambridge, CB, UK), followed by Alexa Fluor 568 (red, Fisher Scientific, Waltham, MA, USA) and then the sections were stained with DAPI (Cell Signaling Technology, Danvers, MA, USA). Images were obtained at 568 nm excitation by a Carl Zeiss LSM800 confocal microscope (Carl Zeiss Microcopy GmbH, Jena, Germany).

Whole-cell lysates were collected in the Laemelli sample buffer as previously described [18 (link)]; nuclear and cytoplasmic fractions were isolated as previously described [19 (link)]. Equal volumes of lysate were blotted and quantified as previously described [2 (link),18 (link),19 (link),20 (link)]. Three pairs of lysates were used to determine the levels of detyrosinated α-tubulin (Abcam, Cambridge, UK), HDAC6, ATAT1 (Abcam), SIRT2 (CST), TGM2 (CST), and HSP90α (CST) expression levels. Whole-cell lysates were collected from BoM-MDA-231 cells following growth under standard culture conditions and blotted. For all western blots conducted using cytoplasmic and whole-cell lysate, β-actin (CST) was used as a loading control. Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA) was utilized as a loading control for western blots involving nuclear protein fractions.