Fig. 3. Exogenous CHMP1 is tightly regulated and enters the nucleus. All experiments use IND-CHMP1 cells and exogenous CHMP1 induction with 10 µM muristerone. (A) Total RNA was harvested at the indicated times of induction and analyzed by northern blot for CHMP1, with CHO-B as an internal control. Endogenous CHMP1 RNA does not comigrate with exogenous and is not detected with this shorter exposure. (B) Cells were harvested at 0 hours and 24 hours of induction, fractionated and analyzed as in Fig. 2. CHMP1 species that comigrate with endogenous (closed arrows) or are novel (open arrow) are indicated. (C) Cells were induced for the indicated times, fixed and double labeled with anti-CHMP1 (red) and Hoechst 33258 (blue). Note the cytoplasmic fibers at 3 hours and 9 hours. (D) Cells were induced for 24 hours, including treatment with colcemid (200 ng/ml) for the final 10 hours to enrich for mitotic cells. Metaphase spreads were prepared (Chandler and Yunis, 1978) and stained for CHMP1 and DNA. The inset shows an enlarged region from the spread. c, cytoplasm; m, nuclear matrix solubilized by detergent; w, whole cell with complete detergent solubilization.