Acetyl α tubulin

Cells were harvested and washed twice with cold PBS after the indicated treatments. Cell pellets were lysed in 30μl of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, and protease inhibitor cocktail) for 30 min on ice. Lysates were centrifuged at 12,000 ×g for 20 min at 4°C, and the protein content in the supernatants was determined using the Bradford reagent (Bio-Rad). Equal amount of protein lysate was resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% non-fat milk powder in PBS-Tween 20 buffer for 1 h, followed by incubation with primary antibody at 4°C overnight. Membranes were washed in PBST and then incubated with HRP-conjugated anti-goat or mouse anti- IgG (Cell Signalling Technology) antibody and detected using the Millipore ECL Western Blot substrate. β-actin was used as a loading control. Antibodies were obtained from the following sources: Acetyl-α- Tubulin (Cell Signalling Technology, 3971S, 1:500), β-actin (Santa Cruz Biotechnology, sc47778, 1:2000), HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1:2000 for β-actin blots, 1:1000 for all others). Blots shown are representative of at least two independent experiments.

MPT0G236 and SAHA (vorinostat) were synthesized by Jing-Ping Liou (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan) at a purity exceeding 98%; the product was previously characterized [22 (link)]. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), sulforhodamine B (SRB), and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO, USA); primary antibodies against acetyl-histone H3, acetyl-α-tubulin, PARP, cleaved caspase 3, caspase 8, caspase 9, cyclin B1, cdc25C, and phosphorylated cdc2 Tyr15 (p-cdc2 (Y15)) were purchased from Cell Signaling Technologies (Beverly, MA, USA); phosphorylated mitotic protein monoclonal 2 (p-MPM2) was purchased from Millipore (Bedford, MA, USA); β-actin was purchased from ABclonal (Woburn, MA, USA); HRP-conjugated anti-mouse and anti-rabbit IgG secondary antibodies were obtained from Jackson ImmunoResearch Inc. (West Grove, PA, USA).

Tissue sections (5 μm) were dewaxed and rehydrated. Antigen retrieval was done by autoclaving the slides in Trilogy solution (Cell Marque, Hot Springs, AR) at 121°C for 10 min. After blocking with 3% H₂O₂ and 5% fetal bovine serum, the slides were allowed to react with against Caspase 3, acetyl-α-tubulin (1:100, Cell Signaling) at 4°C overnight. The slides were then incubated with polymer-HRP reagent (Dako Cytomation, Glostrup, Denmark). The peroxidase activity was visualized with diamino-benzidine tetrahydroxychloride solution (DAKO). The sections were counterstained with hematoxylin. Dark brown nuclear staining was defined as positive and no staining was defined as negative.