Anti aurora b

For Western blotting, cells were lysed in modified RIPA lysis buffer (50 mm Tris–HCl, 150 mm NaCl, 5 mm EDTA, 5 mm EGTA, 0.5% NP-40, 0.5% Triton X–100, 50 mm NaF, 2 mm sodium orthovanadate, 40 mm β-glycerophosphate and 1 μm microcystin) supplemented with a protease inhibitor mix (Thermo Scientific, Rockford, IL, USA). Unless otherwise described, 30 μg of total protein was resolved by SDS–polyacrylamide gel electrophoresis, transferred to filters, and immunoblotted with various antibodies (1:1000 dilution unless otherwise mentioned). The antibodies used were antiAR (sc-7305); antiTPX2; Cdk1 (17 ) and Cdk2 (D-12) all from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-CHIP (C386), anti-Aurora A, anti-pT288-Aurora A, anti-Aurora B and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) from Cell Signaling Technology (Danvers, MA, USA); cyclin B1 (CC03, Calbiochem, Billerica, MA, USA), and mouse monoclonal anti-tubulin antibody from Sigma-Aldrich.

DH/DE-DLBCL cells are lysed in 1X RIPA buffer and supplemented 1:100 Protease/ Phosphatase inhibitor cocktail (Cell Signaling Technology, Danvers, MA). Protein concentrations are determined using the BioRad protein assay kit (Hercules, CA) and 30μg of protein resolved by electrophoresis on a 10% SDS-PAGE. The proteins are transferred onto a nitrocellulose membrane and nonspecific binding is blocked by incubating with 5% nonfat milk in TBS-T buffer (0.01 M Tris–Cl, 0.15 M NaCl, 0.5% Tween-20, pH 8.0) at room temperature for 1h. The membrane is subjected to the indicated antibodies and fluorescence detected using a LI-COR Odyssey Infrared Imaging System. Anti-phospho-Btk (Tyr223) (AB #68217) was purchased from Abcam (Cambridge, MA). C-Myc (SC# 40), Anti-Bcl-2 (SC# 783) antibody was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX). Anti-aurora A (CST #14475) and anti-aurora B (CST #3094), Anti-phospho-aurora A (Thr288) (CST #3079), anti-Akt (CST# 4691), anti-phospho-Akt (Ser473) (CST #4060), anti-phospho-Bcl-2 (CST #anti-2827), Bcl-6 (CST #5650), anti-Bcl-xL (CST # 2762), anti-Btk (CST# 8547), anti-ERK1/2 (CST# 4695), anti-phospho-ERK1/2 (CST# 4370), anti-phospho-Histone H2A.X (CST #5438), anti-PARP (CST #9542) and anti-GAPDH (14C10) (CST #2118), anti-β-Actin (CST# 3700) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

Primary antibodies were diluted in Licor blocking solution/PBST with a final Tween-20 concentration of 0.1% except for anti-phospho Aurora and anti-Histone3 (Ser10p), which had no Tween-20. The following antibodies and antibody dilutions were used: anti-INCENP (raised against C-terminal peptide CSNRHHLAVGYGLKY) (5.5 µg/ml), anti-Aurora B (Kelly et al., 2007 (link)) (5 µg/ml), anti-Borealin A (Dasra A) (Kelly et al., 2007 (link)) (5 µg/ml), anti-Survivin (Tseng et al., 2010 (link)) (12 µg/ml), anti-phospho Aurora (Phospho-Aurora A (Thr288)/Aurora B (Thr 232)/Aurora C (Thr198) 2914, Cell Signaling Technology) (1:200), anti-Histone3 (Ser10p) (6G3, 9706, Cell Signaling Technology) (1:500), anti-Histone3 (T3p) Phospho (Epitomics 2162-1) (1:10000), anti-MBP (New England Biolabs E8032) (1:10000), anti-alpha-tubulin (DM1, Sigma) (1:20000), Incenp (39259, Active Motif) (1:500), AIM-1 (611082, BD Transduction Laboratories) (1:500). Secondary antibodies from Licor were used (Licor goat anti-Rabbit 800 nm and Licor goat anti-mouse 680 nm) as was the Licor imaging system to scan membranes.