Figure 3: Kin-1 localization at centrosomes and its phosphorylation status depend on Plk-1.
From: Kindlin-1 regulates mitotic spindle formation by interacting with integrins and Plk-1
(a) siRNA-induced depletion of Plk-1, but not Cdk5rap2 or Aik-A, resulted in loss of Kin-1 (green) staining from centrosomes. Centrosomes are labelled with anit-γ-tubulin (red). White arrowheads indicate colocalization with Kin-1 (scale bar, 8 μm). (b) siRNA-induced depletion of Plk-1 or treatment of cells with 3 nM BI2536 results in reduced localization of Kin-1 to centrosomes/spindle poles (scale bar, 15 μm). The ‘Colocalization Threshold’ function in FIJI image software was used to determine the extent of colocalization between Kin-1 and Plk-1 (far right panels). (c) Recombinant Plk-1 is able to phosphorylate GST fused to Kin-1 (GST:Kin-1). (d) Two-dimensional SDS–PAGE analysis of Kin-1 reveals up to eight different Kin-1 isoforms (spots 1–8, top blot). Treatment with 15 nM BI2536 for 8 h resulted in the reduction of spot 1 (bottom blot) compared with control (n=2, representative blots shown). Densitometry of the spots was determined using the ‘Plot profile’ feature in FIJI image software and their values plotted and overlaid to more clearly represent the change in the intensity of spot 1 (*) upon BI2536 treatment.
