Anti acetyl α tubulin

Human peripheral blood mononuclear cells (PBMC) (IRB No. CKD-IRB-012) were seeded (1.0 × 10⁶ cells/well) and cultured for 24 hr on 12-well plate before treatment of CKD-506 or LBH-589. The chemicals were treated for 4 hr. After harvesting cells, total protein extracts were prepared. The protein extracts were resolved in pre-made SDS-PAGE (NuPAGE® Bis-Tris Precast Gels, Invitrogen). Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane and probed with the following antibodies; anti-acetyl α tubulin (1:5000, #5335, Cell Signaling Technology), α tubulin (1:2000, #2144, Cell Signaling Technology), anti-acetyl histone H4(1:5000, #8647, Cell Signaling Technology), anti-histone H4(1:1000, #2935S, Cell Signaling Technology), and anti-beta-actin (1:5000, #A5441, Sigma). The horseradish peroxidase-conjugated anti-rabbit IgG (1:5000, #7074S, Cell Signaling Technology) and anti-mouse IgG (1:5000, #7076 S, Cell Signaling Technology) were used as secondary antibodies. Acetylated proteins were visualized by chemiluminescence (RPN2235, GE healthcare) and detected by the Gel documentation system (ChemiDoc™, BioRad).

Cells were lysed in RIPA lysis buffer containing proteinase inhibitors at 4 °C for 20 to 30 min. Protein was quantified using a BCA Kit (Beyotime, China). Protein samples were boiled in 5 × loading buffer, and equal amounts of proteins were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk for 1.5 h and incubated with a primary antibody overnight at 4 °C, the blots were cut prior to hybridization with respective antibodies. The primary antibodies used were as follows: anti-HDAC6, anti-α-tubulin and anti-Acetyl-α-tubulin (Cell Signaling Technology, USA); anti-matrix metallopeptidase MMP2, anti-MMP9, and anti-GAPDH (Abclonal, China). The corresponding horseradish peroxidase-conjugated secondary antibodies were used following primary antibody incubation.

Anti-HDAC6, anti-acetyl-α-tubulin, anti-acetyllysine, anti-Snail, and anti-GSK-3β antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-β-actin, anti-Hsp90, anti-ubiquitin, and protein A/G plus agarose were gained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-N-cadherin was purchased from Thermo Fisher Scientific Inc., (Taipei, Taiwan). Anti-vimentin and anti-E-cadherin antibodies were acquired from GeneTex, Inc. (Irvine, CA, USA) and BD Biosciences, Inc. (San Jose, CA, USA), respectively.