Figure 2.

BCCIPα is the predominant centrosome-associated isoform. (a) The localization of exogenous BCCIP isoforms to spindle poles. HT1080 cells stably expressing YFP-BCCIPα, YFP-BCCIPβ or YFP (negative control) were stained for γ-tubulin (red, centrosomes) and counterstained with DAPI. Illustrated are the representative images at different stages of mitosis. (b) BCCIP has a labile association with centrosomes. Centrosome fractions were pelleted and incubated with the indicated buffers, re-pelleted by centrifugation and the supernatant was collected. The remaining pellet fraction (P) was resuspended in boiling loading buffer and both supernatant and pellet fractions were subjected to SDS-PAGE and western blot. The crude extract (CE) was run concurrently as reference. P: the pelleted centrosomes after buffer treatment; S: the stripped-off proteins in the solution. 1D, 2D, 3D and Urea indicate the buffers with increasing ionic strength (see Materials and Methods). (c) Amino acids 111-257 of BCCIP mediate its localization to spindle poles. The illustrated panel of YFP-tagged full-length and truncated BCCIP proteins were transiently expressed in BCCIP knockdown 293 T cells. Cells were co-probed with α-tubulin to assess spindle pole localization. (d) Expression verification of exogenous YFP-BCCIP fragments. The expression levels of the same panel of BCCIP fragments as in panel-2C verified by western blot.