JCS02922 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Table 10 - Adobe PDF

• Supplemental Table 2 - Adobe PDF

• Supplemental Table 3 - Adobe PDF

• Supplemental Table 4 - Adobe PDF

• Supplemental Table 5 - Adobe PDF

• Supplemental Table 6 - Adobe PDF

• Supplemental Table 7 - Adobe PDF

• Supplemental Table 8 - Adobe PDF

• Supplemental Table 9 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Yeast two-hybrid assays showing an interaction between KIFC5A and Nubp1 and also between Nubp1 and Nubp2. Yeast strain Y190 was co-transformed with the combinations of plasmids indicated. Vector pACT₂ includes the DNA activation domain, vector pAS2.1 the DNA binding domain. Interaction was defined as the ability of over 95% of resulting clones, containing both bait and prey constructs, to grow on triple-selective media (-Leu, -Trp, -His) and also to display b-galactosidase activity upon filter assay. For validation, several negative controls (protein-specific and general), together with positive controls, were devised to exclude non-specific interaction or auto-interaction of each of the three proteins tested.

• Supplemental Figure 2 -

  Fig. S2. Silencing efficiencies achieved for the silencing experiment described in Tables S7-S9 for Nubp1, Nubp2 and the combination of Nubp1and Nubp2 as determined by quantitative real-time PCR. The PBGD housekeeping gene was used for data normalisation.

• Supplemental Figure 3 -

  Fig. S3. Clustering of supernumerary centrosomes (arrows) in KIFC5A-silenced cells. Immunofluorescence for a-tubulin (red), g-tubulin (green) and counterstaining of DNA with Hoechst dye (blue). Bar, 10 mm.

• Supplemental Figure 4 -

  Fig. S4. Atypical cytoplasmic expression of YFP-KIFC5A in a transfected NIH 3T3 fibroblast at interphase. In rare instances, highly overexpressing interphase cells contained YFP-KIFC5A in the cytoplasm, with labelling of the MT network. Since we have not observed this by immunofluorescence, it is not clear whether this corresponds to an artefact of overexpression, or is suggestive of a cytoplasmic pool or the ability of KIFC5A to shuttle. In this image, the nucleus appears ‘overexposed’ in the image because of the extremely high expression of YFP-KIFC5A (green), and labelling extends to the MT network in the cytoplasm, including the centrosome (arrow). DNA is counterstained with Hoechst dye (blue). Bar, 10 mm.