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Figure 5


Fig. 5. The fascin–PKC{gamma} interaction depends specifically on Rac1 activity in migrating cells. (A,B) IKD-F11 Fas–cells cultured in doxycycline were transiently transfected with GFP-Xtfascin and PKC{gamma}-mRFP in conjunction with either N17Rac1-myc or N17Cdc42-myc, and allowed to migrate on 15 nM laminin for 6 hours. Cells were fixed and antibody stained to detect the GTPase and imaged using FLIM to measure FRET. Only N17Rac1 inhibited the FRET interaction. Lifetime images are depicted with a pseudocolour scale where red is a low lifetime (1.75 nseconds) and blue is high (2.4 nseconds). Representative of at least eight cells in each sample. (C) Cumulative FRET efficiency graph for cells migrating on laminin, under control conditions or in cells expressing N17Rac1-myc or N17Cdc42-myc. The mean FRET efficiency for each condition is from seven to nine cells per condition and three independent experiments.





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