Figure 2: Kin-1 is a PCM protein.

(a) Kin-1 localization at centrosomes is absent in cells at G1/S (orange arrowheads) but localizes there as cells enter G2/M (white arrowheads; scale bar, 15 μm). During mitosis, all centrosomes show Kin-1 localization. γ-tubulin is used to mark centrosomes and red arrow heads indicate plasma membrane localization of Kin-1. (b) Western analysis for Kin-1 from lysate of MDA-MB-231 cells arrested at G1/S (0 h) and at time points after release. Kin-1 and Plk-1 levels are higher at G2/M (6 h after release from G1/S arrest) than at G1/S. (c) As cells exit mitosis, Kin-1 and Plk-1 levels are reduced. (d) Costaining of Kin-1 with prominent centrosome markers Plk-1, Cdk5rap-2, Aik-A and NuMA (white arrowheads). Boxed centrosomes in the merged images are enlarged in the bottom panels. Kin-1 (green) and centrosome marker (red; scale bar, 8 μm). (e) Volume rendered three-dimensional presentation of Kin-1 (green) and Plk-1 (magenta) colocalization at centrosomes in a mitotic cell (left; scale bar, 2 μm). Surface rendering of the centrosome immunofluorescence is shown in the right images. (f) Kin-1-specific antibodies were able to coimmunoprecipitate (IP) Plk-1, Aik-A and Cdk5rap2. (g) siRNA-mediated depletion of Kin-1 and Plk-1 results in decreased cell survival as assayed by clonogenic assays. Simultaneous depletion of Kin-1 and Plk-1 results in a further decrease in cell survival compared with depletion of either alone. The percentage of colonies relative to colonies returned with non-targeting siRNA-treated cells is indicated top left.