Nb500 179

The following antibodies were used for immunostaining; Rabbit anti-CtIP (D76F7, #9201, cell signaling, Danvers, MA, USA), mouse anti-CtIP (D-4, 271339, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-TPX2 (NB500-179, NOVUS, Centennial, CO, USA), rabbit anti-γ tubulin (T3559, Sigma, St Louis, MO, USA), mouse anti-α-tubulin (#14-4502-82, Invitrogen), rabbit anti-human ANA-Centromere CREST (90C-CS1058, Fitzgerald, Wicklow, Ireland), mouse anti-BubR1 (ab4637, abcam, Cambridge, UK), rabbit anti-Mad1 (sc-67338, Santa Cruz Biotechnology), rabbit anti-Mad2 (A300-300A, Bethly, Fortis life sciences, Waltham, MA, USA), rabbit anti-phospho Aurora A T288 (3079, Cell Signaling, Danvers, MA, USA), mouse anti-RCC1 (F-2, Santa Cruz Biotechnology). The following antibodies were used for Western blot analysis; mouse anti-CtIP (D-4, 271339, Santa Cruz Biotechnology), rabbit anti-TPX2 (NB500-179, NOVUS), rabbit anti-CyclinB1 (H-433, Santa Cruz Biotechnology), rabbit anti-Histone H3 (06-755, Millipore, Burlington, MA, USA), rabbit anti-phospho-Histone H3 Ser10 (9701, Cell Signaling), mouse anti-β-actin (sc-47778, Santa Cruz Biotechnology), mouse anti-Flag (M2, Sigma) and mouse anti-HA (F-7, Santa Cruz Biotechnology). For Immunoprecipitation assay, mouse anti-Flag (M2, Sigma) and rabbit anti-HA (600-401-384, Rockland, Limerick, PA, USA) were used.

Human or mouse oocytes were fixed in 3.7% paraformaldehyde diluted in PBS for 30 min at room temperature and then permeabilized with 0.2% TritonX‐100 for 15 min. After incubation for 1 h in blocking buffer (1% BSA diluted in PBS with 0.1% TritonX‐100), oocytes were stained with the indicated primary antibodies diluted in blocking buffer overnight at 4°C. After three washes, samples were incubated with Alexa Fluor 568‐conjugated goat anti‐rabbit (A11036, Invitrogen), Alexa Fluor 488‐conjugated goat anti‐rabbit (A11001, Invitrogen), Alexa Fluor 568‐conjugated donkey anti‐mouse (A10037, Invitrogen) secondary antibodies with dilution of 1:300, in combination with 1 µg/ml DAPI (236276, Roche, Basel, Switzerland), with or without FITC‐α‐tubulin (1:400, F2168, Sigma‐Aldrich), or Alexa Fluor 647‐conjugated phalloidin (1:200, A22287, Invitrogen) for 1 h at room temperature. After washing four times, the oocytes were mounted on slides with anti‐fade medium and imaged using a laser‐scanning confocal microscope (LSM800, Carl Zeiss, Jena, Germany). The primary antibodies used were as follows: mouse monoclonal anti‐FLAG (1:500, F1804, Sigma‐Aldrich), rabbit monoclonal anti‐pERK1/2 (1:400, #4370, Cell Signaling Technology, Danvers, MA, USA), and rabbit polyclonal anti‐TPX2 (1:200, NB500‐179, Novus Biologicals, Littleton, CO, USA).