Anti α tubulin dm1α

Primary antibodies used in this study were anti-CDK5RAP2 [78 (link)], anti-TACC3 against aa 126–442 of Gallus gallus TACC3 [78 (link)], anti-phospho-S558-TACC3/P-TACC3 (Cell Signaling), anti-ch-TOG (QED Bioscience, 34032), anti-Clathrin Heavy Chain (Abcam; 21679 and BD Biosciences, 610500), anti-Aurora-A (35C1; Sigma), anti-α-tubulin (Dm1α; Sigma), anti-α-tubulin-FITC (Sigma), anti-γ-tubulin (GTU88; Sigma) anti-phospho-Histone H3 (Millipore), anti-BubR1 (kind gift of W. Earnshaw) and phospho-Aurora-A/B/C (Cell Signaling). For visualization of mitotic spindles and centrosomal proteins, cells were fixed and immunostained as described in [79 (link)]. P-TACC3 antibody staining was carried out in cells fixed in 4% paraformaldehyde (PFA) in PBS. For clathrin antibody, cells were fixed in warm 3% PFA in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl₂ pH 6.8) for 15 mins, followed by extraction with PBS/0.5% TritonX-100 for 15 mins.

Cells plated on glass coverslips were washed in PBS and fixed with 4% formaldehyde for 15 min at room temperature. For centriole staining, cells were fixed with ice-cold methanol at −20°C for 10 min. For E-cadherin staining, cells were fixed with ice-cold 1:1 methanol/acetone at −20°C for 10 min. After fixation, cells were permeabilized in 0.2% Triton X-100 in PBS for 5 min and blocked in blocking buffer (PBS, 5% BSA, and 0.1% Triton X-100) for 30 min. Cells were then stained in primary antibodies diluted in blocking buffer for 60 min. Cells were washed with PBS and incubated with species-specific fluorescent secondary antibodies (Alexa Fluor conjugated; Molecular Probes). DNA was stained with Hoechst 33342 (1:5,000; Invitrogen) for 5 min in PBS. Antibodies used included anti–α-tubulin DM1α (1:1,000; Sigma-Aldrich), anti–centrin-2 N-17-R (1:100; Santa Cruz Biotechnology), anti–γ-tubulin GTU88 (1:500; Sigma-Aldrich), anti–E-cadherin HECD-1 (1:500; Abcam), anti–DDR1 1F10 and 7A9 (1:500; made by B. Leitinger [Carafoli et al., 2012 (link)]), anti-pericentrin (1:1,500; Abcam), anti-HSET (1:1,000; Bethyl Laboratories), anti–β-catenin (1:1,000; Abcam), and anti-p120 (1:1,000; BD Biosciences).