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Fig. 7. (A) Diagram of the KIFC5A-s cDNA used as bait in the yeast two-hybrid system. (B) Protein sequence alignment between Nubp1 and Nubp2. Identical residues are boxed and conservative substitutions are highlighted in grey. The conserved ATP/GTP-binding motif (P loop) is marked in blue, the successive MRP motifs 
and ß are marked in green, and the sequence to which an anti-peptide serum to Nubp2 was produced is marked in orange. (C) In vitro co-selection experiments confirming interactions between KIFC5A&Nubp2, KIFC5A&Nubp1 and Nubp1&Nubp2. In the top panels, bacterially expressed tagged His6-KIFC5A-s, immobilised on Ni2+-NTA agarose beads, was incubated with a lysate from NIH 3T3 fibroblasts expressing GFP-Nubp2 or GFP-Nubp1 (lanes 1), or GFP only (negative control, lanes 2), or GFP-Nubp2 in the absence of pre-bound His6-KIFC5A-s (additional negative control, lanes 3). Bound proteins were probed by immunoblotting with anti-KIFC5A (left panel) or anti-GFP (right panel). A positive signal (detection of GFP-Nubp2 or GFP-Nubp1) is obtained only in the presence of pre-bound His6-KIFC5A-s (no signal in the absence of bound KIFC5A indicates that Nubp2 or Nubp1 does not bind the beads non-specifically). The co-selection of GFP-Nubp2 on the beads is not caused by interaction of KIFC5A with the FP domain (absence of signal in lane 2, right panel). In the bottom panels, the set-up of the experiment and negative controls is similar. GST-Nubp2 was immobilised on glutathione-Sepharose beads and was tested against GFP-Nubp1 (lanes 1), GFP only (negative control, lanes 2) or GFP-Nubp1 in the absence of GST-Nubp2 (additional negative control, lanes 3). Again, the positive signal (detection of Nubp1) was specific only in the presence of GST-Nubp2 (lanes 1). (D) Detection of native Nubp2 in NIH 3T3 cells. Western blot analysis of a total protein extract from NIH 3T3 fibroblasts, using the affinity-purified anti-peptide antibody to Nubp2, revealed a unique band consistent with the predicted Mr of Nubp2. (E) Localisation of Nubp2 in NIH 3T3 cells. Immunofluorescence for Nubp2 (green), centrin (red) and -tubulin (red). DNA was visualised with Hoechst (blue). (E1) Cells in interphase; (E2) cell in prophase; (E3) an aberrant spindle with four asters in prometaphase; (E4) double labelling with anti-centrin and anti-Nubp2 in a cell in prometaphase; (E5) Nubp2 enrichment in centrosomes (arrows) of the fully formed metaphase spindle, shown by double labelling with -tubulin. Bars, 10 µm (E2-E4) and 5 µm (E1). (F) Silencing phenotype after double Nubp1&Nubp2 RNAi, as revealed by triple fluorescence. Cells were immunolabelled for -tubulin, centrin and counterstained for DNA (red, green and blue in the overlay, respectively). Bar, 10 µm.
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