Recombinant Nek2A localizes to centrosomes but not spindle poles. Cell
lines were induced with doxycycline for 24 h before processing for
fluorescence microscopy. GFP-Nek2A (a–c), GFP-Nek2A-K37R (d–f),
Nek2A-myc-His (g–i), and Nek2A-K37R-myc-His (j–l). GFP signal (a
and d), α-myc antibodies (g and j), anti-γ-tubulin antibodies (b,
e, h, and k), and merged image of GFP or myc (green), γ-tubulin (red),
and Hoechst 33258 (blue) (c, f, i, and l). Bar, 15 μm. Centrosomes
(arrowheads) are shown at increased magnification in insets. (B) Merged images
showing Nek2A-K37R-myc-His–induced cells in interphase (a) or metaphase
(b) stained with antibodies against γ-tubulin (red) and myc (green). DNA
is stained in blue. Recombinant Nek2A is detected at interphase centrosomes
(arrowhead) but not mitotic spindle poles (>). Bar, 15 μm. (C) Western
blot with anti-Nek2 antibodies of extracts from cells induced to express
Nek2A-K37R-myc-His (arrowhead) for 24 h before addition of cycloheximide for
the times indicated (hours).