Gtx70017

Oocytes were fixed for 30 min in PBS containing 2% formaldehyde and 0.05% Triton X-100 and then permeabilized in 0.5% Triton X-100 for 20 min at room temperature. Oocytes were incubated at 4 °C overnight in a blocking buffer with 3% bovine serum albumin (B2064, Sigma-Aldrich) in PBS supplemented with 0.1% Tween-20 and 0.01% Triton X-100. Oocytes were incubated at 37 °C with following primary antibodies: anti-β-tubulin antibody (1:200 dilution, ab11309, Abcam), anti-FLAG antibody (1:50 dilution, ab245893, Abcam), anti-centromere antibody (ACA) (1:500 dilution; HCT-0100, ImmunoVision), anti-HEC1 phospho Ser55 antibody (1:200 dilution, GTX70017, GeneTex), anti-BUBR1 antibody (1:100 dilution, 612502, BD Biosciences), and anti-KIF11 antibody (1:200 dilution, HPA010568, Sigma-Aldrich). After washing, oocytes were incubated at 37 °C for 1 h with appropriate secondary antibodies as follows: goat anti-human Alexa Fluor 488 (1:500 dilution, 52526, Sigma-Aldrich), goat anti-rabbit Cy3 (1:500 dilution, AS007, ABclonal), and goat anti-rabbit Alexa Fluor 647 (1:500 dilution, AS060, ABclonal). DNA was counterstained with Hoechst (5 μg/mL, 1:1000 dilution, HY-15559A, MCE) for 10 min at room temperature before imaging. Finally, the oocytes were mounted on a 35-mm glass-bottom dish (801001, NEST) and images were captured using a confocal laser-scanning microscope (Leica SP8 or ZEISS LSM 880).

The following antibodies were used for immunofluorescence and immunoblot: Anti-Mad2 (sc-65492, Santa Cruz Biotechnology, IF 1:75), Bub1 (K0168-3, MBL, IF 1:100, IB 1:1,000), CAP-H (sc-101013, Santa Cruz Biotechnologies, IB 1:1,000), Histone H2A phospho Thr 120^{32 (link)} (IF 1:50), FLAG (012-22384, Wako, IF 1:1,000 or F7425, Sigma, 1:500), SET/TAF1β^{50 (link)} (KM1720, IF 1:50, IB 1:2,000), hSgo2^{51 (link)} (IF 1:2,000 for Fig. 2A and 1:5,000 for Fig. 2F and L, IB 1:1,000), Aurora B (611,082, BD, IF 1:100, IB 1:1,000), pAurora A (T288)/B (T232) (2914S, Cell Signaling, IF 1:50, IB 1:2,000), Hec1 phospho Ser 55 (GTX70017, Gene Tex, IF 1:10,000), cyclin B1 (sc-245, Santa Cruz Biotechnology, IB 1:500), α-tubulin (T9026, Sigma, IF 1:5,000, IB 1:10,000), β-tubulin (T4026, Sigma, IF 1:1,000), GST (sc-459, Santa Cruz Biotechnology, IB 1:1,000) and Cenp-C (PD030, MBL, IF 1:1,000) antibodies. A For immunofluorescence, secondary antibodies (Invitrogen Alexa Fluor) were used at 1:1,000. For immunoblotting, secondary antibodies were used at 1:2,000.

All antibodies were diluted in 3% BSA in PBS. The following primary antibodies were used for immunofluorescence imaging (at the final concentration indicated): chicken α-GFP (ab13970 from Abcam, 1:5000), mouse α-GFP (clone 4E12/8, a gift from P. Parker, 1:1000), rabbit α-pNDC80 Serine 55 (GTX70017 from GeneTex, 1:1000), guinea pig α-Cenp-C (BT20278 from Caltag + Medsystems, 1:5000), rabbit α -BUB1 (A300-373A from Bethyl, 1:1000), mouse α-BUBR1 (A300-373A from Millipore, 1:1000), rabbit α-mCherry (GTX128508, Genetex,1:1000). The rabbit α-pMELT-KNL1 antibody is directed against Thr 943 and Thr 1155 of human KNL1 (Nijenhuis et al., 2014 (link)) (Gift from G.Kops, Hubrecht, NL). The pRVSF-KNL1 (pSer60-KNL1) antibody (custom rabbit polyclonals, gift from I. Cheeseman, MIT, USA) was used at 1:2000 dilution (Nijenhuis et al., 2014 (link)). Secondary antibodies used were highly-cross absorbed goat, α-chicken, α-rabbit, α-mouse or a-guinea pig coupled to Alexa Fluor 488, Alexa Fluor 568, or Alexa Fluor 647 (Thermo Fisher).