Figure 2: NuSAP binds Kid and regulates its localization at microtubules.

(a) NuSAP binds Kid endogenously. Synchronized HeLa cell lysates were incubated with 2 μg rabbit IgG or NuSAP antibody bound to protein A-Sepharose beads. The immunoprecipitated proteins and supernatants were blotted with anti-Kid, anti-NuSAP and anti-GAPDH antibodies. (b) NuSAP binds Kid in vitro. Purified His-NuSAP recombinant protein was incubated with either GST or GST-Kid recombinant protein. GST was pulled down and NuSAP was blotted with an anti-NuSAP antibody. (c) Immunoprecipitation of NuSAP with Kid was conducted with whole-cell lysates of HeLa cells transfected with FLAG-NuSAP after release from thymidine/nocodazole arrest into fresh medium at the indicated time points. The immunoprecipitated protein and the whole-cell lysates were blotted with anti-Kid, anti-FLAG, anti-cyclin B1 and anti-GAPDH antibodies. (d) Kid localization at the kinetochore region in bipolar metaphase HeLa cells and monastrol-treated monopolar cells expressing GFP-NuSAP and the GFP-vector (control). Cells were stained with anti-α-tubulin and Hoechst 33342. The line profile of Kid localization (black) according to α-tubulin (grey) is represented in the right-hand graph. Scale bar, 5 μm. (e) Kid localization in bipolar metaphase HeLa cells transfected with control siRNA or NuSAP siRNA together with GFP-vector or GFP-NuSAP. The line profile of Kid localization (black) is represented in the right-hand graph. Scale bar, 5 μm.