Anti hec1

Cells synchronized with the Cdk1 inhibitor RO-3306 were released with a drug washout and monitored for mitotic entry. Cells were fixed with 4% paraformaldehyde (15 minutes) after ~ 1 hour after drug washout, where the maximal number of cells were observed (by visual inspection) to be in metaphase. Fixed cells were permeabilized in 0.1% Triton X-100 solution for 15 minutes and blocked with 5% goat serum (Invitrogen, Grand Island, NY) for 45 minutes. Primary antibodies were diluted in an antibody dilution buffer consisting of PBS with 1% Bovine Serum Albumin and Triton-X 100 and stored overnight at 4° C. Primary antibodies include Anti-beta tubulin (1:500, mouse monoclonal, 2 28 33, Invitrogen) and Anti-Hec1 (1:1000, human monoclonal) for labeling microtubules and kinetochores respectively. Secondary antibodies diluted in antibody dilution buffer were added along with the conjugated Phalloidin-TRITC (Santa Cruz Biotechnology) or Alexa Fluor 647 Phalloidin (1:40–1:80, Invitrogen) and stored in a dark place for 45 minutes. Secondary antibodies include donkey anti-human IgG Alexa Fluor 555 (1:600) and Goat anti-mouse IgG Alexa Fluor 405 (1:500, Invitrogen). Confocal microscopy was performed using a laser scanning confocal microscope (LSM 880, Carl Zeiss Inc.) with optimal imaging settings and z-slice thicknesses ranging from 0.36–0.5 μm.

Cells were fixed with 4% paraformaldehyde for 15 min. Following 2 times PBS wash, permeabilization was performed with a 0.1% Triton X-100 solution. Permeabilized cells were washed with PBS (2×) and blocked with 5% goat serum (Invitrogen, Grand Island, NY) for 45 min. Primary antibodies were diluted in an antibody dilution buffer consisting of PBS with 1% bovine serum albumin and Triton X-100 and stored overnight at 4 °C. Primary antibodies include anti-β-tubulin (1:500, mouse monoclonal, 2 28 33, Invitrogen), anti-Hec1 (1:1,000, human monoclonal), and anti-phospho-paxillin (1:100, rabbit polyclonal, pTyr31, Invitrogen). Secondary antibodies diluted in antibody dilution buffer were added along with the conjugated Phalloidin-TRITC (Santa Cruz Biotechnology) or Alexa Fluor 647 Phalloidin (1:40 to 1:80, Invitrogen) and stored in a dark place for 45 min. Secondary antibodies include donkey anti-human IgG Alexa Fluor 555 (1:600), goat anti-mouse IgG Alexa Fluor 405 (1:500, Invitrogen), and goat anti-mouse IgG Alexa Fluor 647 (1:500, Invitrogen). Confocal microscopy was performed using a laser scanning confocal microscope (LSM 880, Carl Zeiss Inc.) with optimal imaging settings and z-slice thicknesses ranging from 0.36 to 0.5 µm.