Fig 1: Characterization of HMECsA. Western blotting analysis of tau, KL1, Eg5, and neuron specific beta-III tubulin (βIII-tub) in HMECs. The star denotes big tau and the triangle indicates the normal tau species (Tau). B. Mitotically synchronized cells following nocodazole treatment (N) were comparatively analyzed against unsynchronized control cells (C) for the expression of the same proteins in (A) and also phospho-tau (153T) (pTau (153T)). The control tau and Eg5 bands are as decsribed in (A). The bar graphs show quantification of the protein expression (n=3). The ratios of mitotic protein expression to the control are shown. The expression of protein was normalized to β-actin protein expression. The double asterisks indicate significant differences (Student's t-test, P< 0.05). (C and D) HMECs were immunostained with anti-total (T1029 and Tau13) and phospho-tau (153T) antibodies (green). C. During metaphase and anaphase, tau was found to localize in the spindle. D. Phospho-tau spindle localization (arrowheads) was also detected (a). Mitotic cells (arrowhead) showed stronger signals than interphase cells (b). E. Immunostaining with anti-KL1 (green) antibody detecting a polar KL1 distribution in metaphase and anaphase (a and b). F. Immunostaining for Eg5 (green). In addition to a major broad half spindle distribution of Eg5, “static Eg5” (arrowhead) was detected in the middle of the spindle. G. Immunostaining for cytoplasmic dynein (green), γ-tubulin (magenta), and tubulin (red). H. Molar ratio of tau to tubulin heterodimers on the mitotic spindle. Polymerized MTs (P fraction) were purified from mitotic HMECs and assessed for total tau and tubulin by immunoblotting. GST-tagged proteins were used as standards. Bars, 5μm.