Figure 4.
hSpindly targets both dynein and dynactin to KTs. (A) HeLa S3 cells were treated with GL2 or hSpindly siRNAs for 48 h. Nocodazole was added to the cells 2 h before they were stained with CREST serum (green), anti-p150Glued antibody (red), and DNA (blue). (B) Cells treated as in A were stained with CREST serum (green) and anti-dynein intermediate chain (DIC) antibody (red) and DNA (blue). (C) Cells were treated with GL2, hSpindly-1, or hSpindly-2 siRNAs for 48 h. Lysates from mitotic cells (nocodazole shake-off) were prepared and equal amounts of cell extracts were separated by SDS-PAGE and probed by Western blotting with the indicated antibodies. (D) Cells were transfected with hSpindly-2 siRNA together with either a myc-vector construct (V) or a myc-tagged hSpindly (siRNA-resistant) construct (WT) for 48 h. Cells were stained with anti-myc, anti-p150Glued antibodies (red), CREST serum (green), and DAPI (blue). (E) Cells were treated as in D. Lysates were prepared and equal amounts of cell extracts were separated by SDS-PAGE and probed by Western blotting with the indicated antibodies. (F) Table showing positive/negative localization of the indicated KT/centromere proteins after hSpindly depletion for 48 h (−, lost from KT; +, indistinguishable from control). For DIC and p150, experiments were repeated with treatment of nocodazole (2 h) to confirm their dependency on hSpindly. Bars, 10 µm.