α tubulin dm1α

Primary antibodies for immunoblotting were FLAG (M2) from Sigma Aldrich; STAG2 (J-12; carboxyl-terminus epitope), STAG1 (A-9), RAD21 (B-2), SMC3 (E-3), SMC1A (M-16), WAPL (A-7), and PDS5A (S-20) from Santa Cruz Biotechnology; and PDS5B (A300-537A) and STAG2 (A302-580A; amino terminus epitope) from Bethyl Laboratories. Antibodies and conjugated beads for immunoprecipitation were FLAG-M2 beads from Sigma Aldrich, SMC3 (A300-060A) from Bethyl Laboratories, and Streptavidin Plus UltraLink Resin from Pierce. Antibodies for immunofluorescence were anti-centromere antibody (ACA) from Geisel School of Medicine, α-tubulin (DM1α) from Sigma Aldrich, and Alexa Fluor 488 and 568 from Molecular Probes.

After drug treatment, cells were trypsinized, washed once with PBS, centrifuged at 500 rpm for 5 min using a cytospin (Thermo Fisher Scientific, Waltham, MA) and seeded onto poly-l-lysine (PLL)-coated coverslips. The cells were then fixed with 4% para-formaldehyde for 20 min, blocked in 2% BSA/PBS for 30 min, and incubated in primary anti-pH3 and anti-γH2AX antibody overnight (4°C). Cells were then washed three times in 2% BSA/PBS, incubated in Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies for 1 hour at room temperature. DAPI was added to stain nuclei. Cells were imaged on an upright fluorescence microscope (Nikon, Melville, NY), and the data was analyzed using FociCounter (Anna Jucha, University of Wrocław, Poland). Cells were scored based on whether they harbor ≥10 γH2AX foci.
For multipolar and monopolar mitotic spindles staining, cells were washed with PBS and fixed with cold methanol for 20 min at −20°C, followed by incubation with primary antibodiesmin including Cep192 (SPD-2, A. Dammermann, K. Oegema Lab) and α-tubulin (DM1α, Sigma). Images were recorded on a Deltavision microscope at 1 × 1 binning with a 100× NA 1.3 U-planApo objective. Z-stacks (0.2 μm sections) were deconvolved using softWorRx (Applied Precision) and maximum intensity projections were imported into Adobe Photoshop CS4 (Adobe) for analysis.