Pt288 aurora a

The cells were harvested and lysed using RIPA lysis and extraction buffer (GeneDepot, Katy, TX, USA), 1% phosphatase inhibitor (GeneDepot) and 1% protease inhibitor cocktail (GeneDepot) on ice for 10 min, and debris was removed by centrifugation. The proteins were separated using 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Amersham^TM Hybond^TM, GE Healthcare, Chicago, IL, USA). The membranes were blocked in TBS-T containing 3% BSA for 1 h at room temperature and then incubated with primary antibodies against β-actin (1:5000, 4967S, Cell Signaling Technology), cyclin B1 (1:1000, 4138, Cell Signaling Technology), p-MPM2 (1:1000, 05-368, e, Sigma, Burlington, MA, USA), pT288-Aurora A (1:1000, 3079S, Cell Signaling Technology, Danvers, MA, USA), cleaved-PARP (1:1000, 9541S Cell Signaling Technology), or pY15-Cdk1 (1:1000, 9111, Cell Signaling Technology) overnight at 4 °C. After washing thrice, the membranes were incubated with the appropriate HRP-conjugated anti-rabbit (1:2500, GeneDepot) or anti-mouse (1:2500, GeneDepot) secondary antibodies for 30 min at room temperature. The HRP-conjugated secondary antibody was detected using an enhanced chemiluminescence detection system (Amersham Life Science, Piscataway, NJ, USA), and the bands were imaged using an Amersham Imager 600 system (GE Healthcare).

Primary antibodies for 14-3-3γ (CG31; Abcam ab76525; dilution 1:2500), 14-3-3ε (T16; Santacruz sc1020; dilution 1:1000), 14-3-3σ (CS112 tissue culture supernatant 1:50), β-actin (Sigma A5316; dilution 1:5000), GFP (Clontech 632375; dilution 1:15,000), NPM1 (Invitrogen 325200; dilution 1:5000), phospho-T199 NPM1 (Abcam ab81551; dilution 1:2000), Aurora A (Invitrogen 458900, dilution 1:1000), p-T288 Aurora A (Cell signaling technology 3079, dilution 1:1000) and h-Sas6 (110 (link) dilution 1:3000) were used for Western blot experiments. The secondary goat anti-mouse HRP (Pierce) and goat anti-rabbit HRP (Pierce) antibodies were used at a dilution of 1:2500 for Western blot analysis. Primary antibodies for Cep-170 (Invitrogen 41-3200; dilution 1:50), γ-Tubulin (Sigma T3559; dilution 1:200), α-tubulin (Abcam ab7291; dilution 1:500), centrin1 (Abcam ab11257; dilution 1:50), Ninein (Abcam ab4447; dilution 1:50), 14-3-3γ (CG31; Abcam ab76525; dilution 1:200) antibodies were used for immunofluorescence. Secondary antibodies (conjugated with Alexafluor-568, Alexafluor-546, Alexafluor-455 from Molecular probes, Invitrogen; dilution 1:100) were used for immunofluorescence studies.

Cells were lysed using NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.5% Nonidet P-40, 50 mM β-glycerophosphate, 10 mM NaF and 1 mM Na₃VO₄) with protease inhibitor (Millipore, 535140) on ice for 10 min. After centrifugation at 12,000 ×g for 5 min, the supernatant was saved as a crude cell extract. The crude cell extracts were boiled in the Laemmli buffer and then loaded onto a SDS-polyacrylamide gel [68 (link)]. The antibodies used for Western blotting are as follows: PARP-1 (Santa Cruz Biotechnology, sc-7150), p62/SQSTM1 (Cell signaling, 5114), LC3 (MBL International, PM036), Cyclin A (Santa Cruz Biotechnology, sc-751), Cyclin B1 (Santa Cruz Biotechnology, sc-752), Cyclin D1 (Santa Cruz Biotechnology, sc-753), p53 (Santa Cruz Biotechnology, sc-126), p21 (EMD Millipore, OP64), Aurora A-pT288 (Cell Signaling, 3079), Aurora A (Cell Signaling, 4718), Aurora A-pT288/Aurora B-pT232/Aurora C-pT198 (Cell Signaling, 2914), Aurora B (Cell Signaling, 3094), PLK1-pT210 (Santa Cruz Biotechnology, sc-135706), PLK1 (Cell Signaling, 4513), H3-pS10 (Upstate, 06-570), β-actin (Cell Signaling, 4970), and GAPDH (Santa Cruz Biotechnology, sc-25778).