FIG. 5.

Displacement of centrosomal Nlp by Plk1 is blocked by kinase-inactive Nek2A. (A) Human U2OS cells were cotransfected for 24 h with Myc-tagged Nek2A, Nek2A-K37R (K/R), Plk1-T210D (T/D), or Plk1-K82R (K/R), as indicated, before methanol fixation and processing by immunofluorescence microscopy with appropriate antibodies to detect transfected proteins (α-Myc) and endogenous Nlp. The percentage of cells with reduced or absent Nlp at interphase centrosomes was calculated in >100 cells for each sample in three independent experiments. Western blots show the level of expression of the transfected proteins (Myc), and equal loading was confirmed with an antitubulin blot. (B) Transient transfections were performed and analyzed as in A except that after only 16 h of transfection. (C) Representative images of endogenous Nlp (b and d) in cells transfected with Plk1-T/D alone (a and b) or in combination with Nek2A-K/R (c and d) are shown. Scale bar, 10 μm.