Mouse anti mad2

For staining of kinetochore proteins, cells on coverslips were pre-extracted with PHEM-buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO4) supplemented with 0.5 % Triton-X-100 for 15 min and after that fixed with 2 % paraformaldehyde in 0.5 % Triton-X-100/PHEM for 15 min. For the whole cell analysis, cells were fixed directly with 2 % paraformaldehyde in 0.5 % Triton-X-100/PHEM for 15 min and then rinsed with MBST (10 mM MOPS, 150 mM NaCl and 0.05% Tween 20). Next the cells were blocked with 20 % boiled normal goat serum (bngs) in MBST for 1h at RT, followed by staining with primary antibodies for overnight at 4°C or 1h at RT. Primary antibodies used were mouse anti-Mad2 (Santa Cruz, Dallas, TX, USA, sc-65492, 1:75), anti-Bub1 (Abcam, ab9000, 1:150) and human autoimmune serum (Crest, Antibodies Incorporated, Davis, USA, 1:200). The secondary antibody dilutions and sample mounting were performed as described elsewhere [51 (link)].

The primary and secondary antibodies were purchased from commercial sources as follows: Mouse anti-FTO, Mouse anti-Mad2, Mouse anti-Cdc20, Mouse anti-Bub1, Mouse anti-Bub1b, Mouse anti-Bub3, Mouse anti Tubulin (Santa Cruz Biotechnology), Rabbit anti m⁶A (Synaptic Systems), Rabbit anti-Actin (Sigma-Aldrich). HRP-goat anti rabbit IgG (CWbio) and HRP-goat anti mouse IgG (CWbio).