Hdac6

Cell lysates were prepared using a buffer containing 10 mmol/L Tris-HCl at pH 7.4 and 1% sodium dodecyl sulfate (SDS). Protein concentration was determined using the Pierce Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher Scientific). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was then performed on the cell lysates, transferred to polyvinylidene fluoride membranes, and immunostained overnight using antibodies as described below. The following antibodies were purchased from Cell Signaling Technology: p21 (dilution at 1:1,000), p27 (1:1,000), cyclinB1 (1:1,000), aurora kinase A (AURKA; 1:1,000), polo-like kinase 1 (PLK1; 1:1,000), cyclin-dependent kinase 1 (CDK1; 1:1,000), E-cadherin (1:1,000), Vimentin (1:1,000), acetyl-histone H3 (1:1,000), histone H3 (1:1,000), p44/p42 MAPK (ERK1/2; 1:1,000), p-p44/p42 MAPK (ERK1/2; 1:1,000), p-AKT1 (1:1,000), and AKT1 (1:1,000). Aurora kinase B (AURKB; 1:1,000) and HDAC2 (1:1,000) antibodies were purchased from Abcam. Survivin (1:1,000) was obtained from Novus Biologicals, N-cadherin (1:1,000) from EMD Millipore, HDAC1 (1:1,000) from Thermo Fisher Scientific, and HDAC10 from BioVision. The following antibodies were purchased from Santa Cruz Biotechnology: p27(1:1,000), HDAC4(1:1,000), HDAC6 (1:1,000), and GAPDH (1:5,000). Band densitometry analysis was performed using ImageJ software (NCI).

For EB1 protein detection, media was removed and the cells were immediately
fixed in −20°C methanol (Fisher Scientific, Pittsburgh, PA) for 5min. For
all other stains, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, St.
Louis, MO) prepared in phosphate buffered saline (PBS) for 30min at room temperature.
Cells were permeabilized with 0.1% Triton X-100 (Fisher Scientific, Pittsburgh,
PA) in PBS for 20min and then blocked with a commercial blocking buffer (SuperBlock,
Thermo Scientific, Rockford, IL) for 1hr at room. Cells were incubated in primary antibody
overnight at 4°C or 1hr at room temperature. Secondary antibody was used at a
dilution of 1:1000 for 1hr at room temperature. Both primary and secondary antibodies were
diluted in a commercial antibody diluent (BioGenex, Fremont, CA). Primary antibodies used:
α-tubulin (1:5000; Sigma-Aldrich, St. Louis, MO), acetylated-tubulin (1:1000;
Sigma-Aldrich, St. Louis, MO), EB1 (1:500; BD Biosciences, San Jose, CA), FLAG (1:5000;
Sigma-Aldrich, St. Louis, MO), HDAC6 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA),
and SIRT2 (1:1000; Sigma-Aldrich, St. Louis, MO). Secondary antibodies used: Alexa
Fluor-488 (Invitrogen, Carlsbad, CA) and Cy3 (Jackson ImmunoResearch Laboratories, West
Grove, PA).