Anti β tubulin h 235

Primary antibodies: anti-YB-1 raised against the region 1 to 100 of YB-1 protein (12148 Abcam, Cambridge, UK); anti-PABP1 (poly(A)-binding-proteiin 1) (clone 10E10, Sigma-Aldrich); anti-Myc (SAB21084786, Sigma-Aldrich, Saint Louis, MI, USA); anti-cyclin D1 (GT8912, Genetex, Irvine, CA, USA); anti-cyclin A2 (GT2547, Genetex, Irvine, CA, USA); anti-GAPDH (6C5 Santa Cruz Biotechnology, Dallas, TX, USA); anti-PARP1 (Cell Signalling, Danvers, MA, USA); anti-RNH1 (436–450, Sigma-Aldrich, Saint Louis, MO, USA) anti-β-tubulin (H-235, Santa Cruz Biotechnology, Dallas, TX, USA); anti-actinin (AT6/172, Abcam, Cambridge, UK); anti-p21 WAF (1D21 Cell Signaling, Danvers, MA, USA); anti-∆Np63α (4A4) (sc-8431 Santa Cruz Biotechnology, Dallas, TX, USA).
Secondary fluorescent antibodies: Alexa Fluor 488 anti-rabbit (Thermo-Fisher Scientific, Waltham, MA, USA); Cy3 anti-mouse (Jackson ImmunoResearch, Cambridge, UK); DAPI (Sigma-Aldrich, Saint Louis, MO, USA).

Cells were lysed in cell lysis buffer (50 mM Tris-HCl, pH7.5, 120 mM NaCl, 1 mM EDTA, and 1% NP-40) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail 2 (Sigma). Cellular lysates (1 mg) were immunoprecipitated, using the anti-CDC20 (5 μl, AR12, Millipore, Burlington, MA, USA) or anti-MAD2 (5 μg, A300, Bethyl Lab, Montgomery, TX, USA) antibodies. The immunoprecipitated products or total lysates were resolved on SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes (BioRad). Proteins were detected with specific antibodies. The following antibodies were used in western blot analyses: anti-Cyclin B1 (H433), anti- CDC2 [54 (link)], anti-β-Tubulin (H-235, Santa Cruz Biotechnology, Santa Cruz, CA, USA); ant-MAD2 [48 (link)], anti-phosphoserine/threonine (22A, BD Biosciences); anti-β-Actin (M2, Sigma); anti-cyclin A2 (BF683), anti-cleaved Caspase 3 (D175), anti-p-RPS6 (S235/236) (2211), anti-p-CDC2 (Y15), anti-RB (4H1), anti-p-RB (S780) (D59B7), anti-APC2 (12301, Cell Signaling Technology, Danvers, MA, USA); anti-MPM2 (Millipore); anti-CDC20 (A301, Bethyl Lab). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Cell Signaling Technology; GeneTex, Irvine, CA, USA) were used and proteins were visualized with an enhanced chemiluminescence system (Advansta, Menlo Park, CA, USA).