Table 1.

Fluorescence recovery after photobleaching analysis of Nek2A

Protein	Treatment	Bleach area	Centrosome	Half-life (t1/2)	% Recovery
GFP-Nek2A		50 × 50	Paired	3.2 s	73
GFP-Nek2A-K/R		50 × 50	Paired	3.4 s	64
GFP-Nek2A	+ Nocodazole (7.5 μg/ml)	50 × 50	Paired	3.5 s	78
GFP-Nek2A		50 × 50	Split	3.0 s	65
GFP-Nek2A	+ Nocodazole (7.5 μg/ml)	50 × 50	Split	3.2 s	49
GFP-Nek2A		250 × 250	Paired	12.5 s	25
GFP-Nek2A	+ Nocodazole (7.5 μg/ml)	250 × 250	Paired	19.5 s	18
GFP-Nek2A		250 × 250	Split	12.0 s	35
GFP-Nek2A	+ nocodazole (7.5 μg/ml)	250 × 250	Split	21.3 s	25
GFP-Nek2A		Spot	Split	2.8 s	63
GFP-Nek2A	+ Nocodazole (7.5 μg/ml)	Spot	Split	3.0 s	62
Centrin-GFP		50 × 50	Paired	>5 min	nd

The half-life and % fluorescence recovery at the centrosome was determined for GFP-Nek2A, GFP-Nek2A-K37R, and centrin-GFP proteins with or without drug treatment and with small or large bleach area as indicated. The Nek2A constructs were induced for 24 h with tetracycline in the relevant U2OS cell line, whereas the centrin-GFP was constitutively expressed in a stable CHO cell line. Photobleaching and subsequent confocal imaging was performed as described in Materials and Methods. The rate of recovery of centrin-GFP was so slow that the final % recovery could not be determined (nd) within the time frame of the experiment.