Fig. 6. Constitutively active Rac1 or PKC, but not Cdc42, activates the interaction of fascin and PKC in quiescent cells. (A) Lifetime of cells expressing GFP-Xtfascin alone. (B) IKD-F11 Fas–cells were transiently transfected with GFP-Xtfascin and the indicated mRFP acceptor constructs in the absence or presence of constitutively active Rac1 or Cdc42. Quiescent, serum-starved cells were fixed and antibody-stained to detect the GTPase and imaged using FLIM to detect FRET. Lifetime images are depicted with a pseudocolour scale where red is a low lifetime (1.85 nseconds) and blue is high (2.4 nseconds). FRET was increased in the presence of V12Rac1-HA (arrow indicates examples of strong interaction in filopodia) or PKCA25E-mRFP, but not in the presence of V12Cdc42-HA. (C) Cumulative FRET efficiency graph for control cells or cells expressing the indicated constructs. The mean FRET efficiency for each condition is from seven to nine cells per condition and three independent experiments.