Figure 1 - HEF1 localization to the centrosome: cell-cycle and sequence dependence.

(a) Cell-cycle-synchronized populations were analysed for HEF1 localization to the centrosome and mitotic apparatus. HEF1 is indicated in green, and visualized with anti-HEF1-SB-R1 antibodies, as previously described³. -tubulin (red) is used to indicate centrosomes (arrows). DNA (blue) becomes condensed at mitotic entry. Enlarged views of boxed centrosomes are shown in the bottom right corners; arrows mark centrosomal location. Images are merged confocal sections. Scale bar of 8 m applies to top row; 6 m scale bar applies to all other images. (b) FACS analysis demonstrating asynchronous (Asy.) MCF-7 cells, and MCF-7 cells synchronized in G1, S and G2/M phases, as used for immunofluorescence analysis. (c) Western analysis of HEF1 levels in the indicated phases of the cell cycle, with -actin as loading control. The HEF1 doublet represents two phosphorylation-induced isoforms of HEF1 with relative molecular masses of 105,000 (M_r 105K) and 115,000 (M_r 115K) (hyperphosphorylated)¹⁰. The broad band migrating at 95K is a non-specific, cross-reacting species detected with the rabbit polyclonal antibody, described in ref. 10 (also see Supplementary Information, Fig. S1A). (d) Cells transfected with plasmids expressing GFP–HEF1_1–363 or GFP–HEF1_1–405, and stained with antibody to -tubulin (red). White lines in lower panels outline cell peripheries. Boxed centrosomes are enlarged in insets; arrows indicate other examples. (e) Fragments of HEF1 analysed as GFP- or FLAG epitope-tagged fusion proteins, amino acids and the degree of localization to the centrosome are shown. The degree of centrosomal localization was estimated by measuring the signal intensity at the centrosome in the same set of experiments (data not shown). HEF1_1–834 is full-length HEF1. Asterisk, protein poorly expressed.