Fig. 2.
Recruitment of Alix and ESCRT-III to the midbody is essential for normal midbody morphology. (A) HeLa cells stably transduced with retroviral vectors encoding mCh or mCh-AlixR fusions were treated with siRNA targeting Luciferase or Alix, and resolved cell lysates were analyzed with α-Alix or α-Hsp90 antibodies. Alternatively, cells were fixed, stained with α-tubulin, and scored for aberrant midbodies (n = 3 ±SD) and multinucleation (n > 4 ±SD). (B) Representative micrographs of aberrant midbodies from A, enlargement depicting α-tubulin channel. (Scale bars: 10 μm.) (C) Clonal HeLa cells stably expressing YFP-Cep55 were treated with siRNA targeting Luciferase or Alix. Cells were fixed and stained with α-tubulin. Enlargements depict the α-tubulin channel. (Scale bars: 10 μm.) Alternatively, cells were lysed and analyzed with α-Alix, α-Cep55, and α-Hsp90 antisera.