Mouse anti aurora b

For immunoprecipitation, cells were lysed using a sonicator (EpiShear Probe Sonicator, Active motif, 120 W, 20 KHz) in E1A buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% NP-40, 1 mM dithiothreitol, 5 mM EDTA) containing protease and phosphatase inhibitors (1 mg ml⁻¹ aprotinin, 1 mg ml⁻¹ leupeptin, 5 mM NaF and 0.5 mM Na₃VO₄). Protein lysates were immunoprecipitated with indicated antibodies for 12 h at 4 °C under constant rotation. The protein–antibody complex was further incubated with protein A-Sepharose beads (GE Helathcare) for 1 h 30 min at 4 °C and the immune complex were washed and subjected to immunoblotting. For immunoblotting, the following antibodies were used: mouse anti-RSF1 (Upstate, 05-727); rabbit anti-PLK1 (Santa Cruz Biotechnology, sc5585) and mouse anti-Aurora B (BD science, 611083); mouse anti-Flag (M2 Flag sigma, F3165); mouse-Myc (Santa Cruz Biotechnology, sc-40); and mouse HA (Santa Cruz Biotechnology, sc-7392) antibodies. For in vitro binding assays, Flag-PLK1, Myc-PLK1 and HA-Aurora B proteins were immunopurified from mitosis-enriched HEK293T cells. Recombinant proteins of His-PLK1, GST-PLK1, GST-RSF1 and RSF1-V5 were prepared as described in the above. The proteins were incubated for 2 h at 4 °C under constant rotation and beads-bound immune complexes were washed for four times and subjected to immunoblot analysis.

Cell lines were seeded into 6 wells plates and synchronised and released from a thymidine block into medium supplemented with 20 μM STLC. Seven hours later cells were harvested and washed with PBS once followed by lysis in Laemmli buffer. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting were performed using the standard Bio-Rad protocols. The nitrocellulose membranes were blocked in TBS/0.1% Tween 20 (TBST) containing 4% milk for 30 min and incubated with the following primary antibodies: mouse anti-INCENP (1:500, Invitrogen—39–2800), mouse anti-Aurora B (1:250, BD Transduction Labs—611,083), mouse anti-α-tubulin (1:10,000, Sigma—T5168), or rabbit anti-GFP (custom made). After washing in TBST, the membranes were incubated in HRP-conjugated secondary antibodies goat anti-mouse (1:2500, Dako—P0447) or goat anti-rabbit (1:2500, Dako—P0448) in TBST-4% milk ECL (Advansta) was used as substrate of HRP and chemiluminescence was measured using an Amersham Imager 600. Uncropped scans of western blots can be found in Supplementary Fig. 7).