Fig. 2.
FRAP reveals mobilization of SMN from Cajal bodies by FGF-223. For live cell imaging, single CBs of pSMN-EGFP and pFGF-223-DsRed2 or pFGF-218-DsRed2 or pDsRed2 coexpressing HEK293T cells were bleached for 1 s (405 nm). Fluorescence recovery was measured 10 s before and 10 s after photobleaching and then every minute up to 20 min unless the focused nuclear body left the volume as defined by z-sections. a Example of a single FRAP measurement. Fitting of a one-exponential curve is represented by the black line; fitting of a two-exponential curve is shown by the green line. Data display best fit to a two-exponential model. b Example of a SMN-EGFP and FGF-223-DsRed2 coexpressing cell before and after photobleaching. Scale bar 10 μm. c Summary kinetics of averaged FRAP measurements (n = 10). FGF-223-DsRed2 increased the fluorescence recovery of SMN-EGFP to CBs. d The immobile populations of SMN-EGFP was significantly decreased after overexpression of FGF-223-DsRed2. The fast population of SMN-EGFP was significantly increased compared to the DsRed2 control. The slow populations were not significantly altered. The recovery halftimes (t 1/2) of the slow and fast populations of SMN-EGFP were non-significantly changed after overexpression of FGF-223-DsRed2 (means ± SEM; *p < 0.05; n.s. non-significant; unpaired, two-tailed t test)