α tubulin dm1a

Striatum tissues were homogenized with 10 volumes of sucrose buffer (0.32 M sucrose, 1 mM NaHCO3, 1 mM MgCl2, and 0.5 mM CaCl2, plus protease and phosphatase inhibitor cocktails) and centrifuged at 10,000 g for 10 min. Protein concentrations in supernatant were measured by BCA (Thermo Fisher Scientific). Proteins were size–fractioned by 4–12 % NuPage BisTris– polyacrylamide gel electrophoresis (Life Technologies) using MES running buffer (Life Technologies). After transfer to nitrocellulose membranes, the membranes were immunoblotted with the appropriate dilutions of the primary antibody: human α–syn (syn211, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) and α–tubulin (DM1A, 1:2000; Santa Cruz Biotechnology, Santa Cruz, CA. Signals were visualized with fluorescent secondary antibodies and quantified with ImageJ.

Western blot analyses of total protein extracts were performed as previously described [12 (link)]. Immunodetection was performed using antibodies directed to: H3 histone (abcam), H3 acetylated histone (Millipore, Billerica, MA, USA), α-tubulin (DM1A) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-α-tubulin (K40) (Sigma-Aldrich), γH2AX (Ser139) (Millipore), PARP cleaved (Millipore), PARP (Santa Cruz Biotechnology) β-actin (Sigma-Aldrich), HSP72/73 (Calbiochem, San Diego, CA, USA), anti-mouse or anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Cell Signaling; Amersham Biosciences, Freiburg, Germany). Antibody binding was visualized by enhanced chemiluminescence method (Amersham Biosciences) according to manufacturer's specification and recorded on autoradiographic film (Amersham Biosciences). Densitometric evaluation was performed using Image J software and normalized with relative controls depending on the Analysis.