Figure 2: Hyperphosphorylation of EB2 during SAC-dependent arrest.

(a) Immunoblot analysis of EB2 in asynchronous (Asyn.), taxol-arrested, nocodazole-arrested (Noc.) and monastrol-arrested (Mona.) extracts from whole or shake-off HeLa cells. (b,c) HeLa cells were synchronized by a double-thymidine block and then nocodazole was added 1.5 h after release (b), or MG132 was added 6.5 h after release (c). See Supplementary Fig. 9b for experimental scheme. (d) HeLa cells were synchronized by a single thymidine block and then after 3 h released into nocodazole for 11 h. Synchronized HeLa cells were harvested by mitotic shake-off and transferred into fresh medium with or without nocodazole. See Supplementary Fig. 9c for experimental scheme. (e) MT co-sedimentation assay. Asynchronous and monastrol-arrested HeLa cells were lysed, pre-cleared (HSS) and subjected to a MT co-sedimentation assay. HSS was incubated for 30 min with exogenous purified porcine brain tubulin that was either taxol-stabilized at 37 °C (WP and WS fractions) or colchicine-depolymerized on ice (CP and CS fractions). The samples were centrifuged at 100,000 g to separate the MT polymers (WP and CP) from the soluble tubulin dimers (WS and CS) and immunoblot analysis was carried out to assess the presence of EB2 and tubulin. See Supplementary Fig. 9d for experimental scheme. CP, cold pellet; CS, cold supernatant; HSS, high-speed supernatant; WP, warm pellet; WS, warm supernatant. Immunoblot analysis was carried out using antibodies against the indicated proteins.