Supplementary Figure 3: Kif25 MT binding in vitro and in centrosome separation.

(a) MT cross-linking by EGFP-Kif25 was shown in vitro using Alexa-568 labeled taxol stabilized MTs and 500 nM of conventional kinesin I or EGFP-Kif25, scale bar, 20 μm Shown are representative images from 3 experiments. (b) Asynchronous HeLa cells were fixed and stained for α- and γ-tubulin before imaging interphase cells for centrosome separation. Cumulative frequency plot shows data pooled from 3 separate experiments in each condition (n = 150 control, n = 150 Kif25 KD Noc cells). Control or Kif25 overexpressing cells were treated with 20 μM nocodazole for 2 hr prior to fixation to depolymerize MTs. (c) Schematic of kinase signaling cascade leading to centrosome separation. (d) Quantitative IF to determine the relative levels of phosphor-Aurora A, Phospho-Plk1, Nek2A, and C-Nap1 at the centrosome during interphase. n = 75 cells were analyzed in each condition in cells stained with the indicated antibody and γ-tubulin to mark the centrosome. A circular ROI was drawn around each centrosome and the intensity measured for each experimental protein. A second intensity measurement was taken in the cytoplasm as background intensity for each cell. The displayed intensity represents the background corrected value for protein. Error bars represent mean ± s.e.m., ^∗P = 0.03, ^∗∗P = 0.0015. N. S; not significant. Statistics were analysed using an unpaired t-test.