Western blotting and co-immunoprecipitation

Western blot analysis was performed as previously described (van Ree et al., 2010). Lung tissue lysates were prepared as previously described (Baker et al., 2013). Briefly, the lung tissue was snap-frozen in liquid nitrogen and then ground into powder with a mortar and pestle. Ten milligrams of the powder was resuspended in 100 μl of PBS, boiled for 10 min at 100°C after the addition of 100 μl Laemmli lysis buffer (Bio-Rad, Hercules, CA), and loaded into Tris-HCl polyacrylamide gels (Bio-Rad). Primary antibodies used were mouse anti-BubR1 (BD Transduction, San Jose, CA; 612503, 1:1,000), rabbit anti-mouse BubR1 (aa382-420) ([Baker et al., 2004]; 1:1000), rabbit anti-human BubR1 (aa1-350) ([Baker et al., 2004]; 1:1000), rabbit anti-Flag (Sigma-Aldrich, St. Louis, MO; F7425, 1:1000), rabbit anti-Flag (Cell Signaling, Danvers, MA; 2368S, 1:1000), rabbit anti-Cdc20 (Santa Cruz, Dallas, TX; sc-8358, 1:1000), mouse anti-Kras (Santa Cruz; sc-30, 1:1000) and rabbit anti-pCdc20^S92 and rabbit anti-pCdc20^S153 (generous gifts from Hongtau Yu). All antibodies were detected with secondary HRP-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch, West Grove, PA; 1:10,000). Ponceau S staining (1% glacial acetic acid, 1.1 g/ml Ponceau S [Sigma-Aldrich]) served as a loading control for blots. All western data are representative for two or three independent experiments. Co-IP was performed with mitotic MEFs that were immortalized by expression of SV40 large T antigen as previously described (Baker et al., 2013). Primary antibodies used were mouse anti-BubR1 (BD Transduction; as above), rabbit anti-mouse BubR1 (aa382-420) ([Baker et al., 2004]; as above), rabbit anti-Cdc20 (Santa Cruz; as above), mouse anti-Mad2 (BD Transduction, 610679, 1:1000), rabbit anti-Mad2 ([Ricke et al., 2011]; 1:1000). All antibodies were detected with secondary HRP-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch; as above) except when Cdc20 immunoblot was performed from CDC20 IP, in which Rabbit TrueBlot Anti-Rabbit IgG HRP (Rockland, Limerick, PA; 18-8816-33 1:1000) was used.