Fig. 7. Role of Pak1 in regulating the fascin–PKC interaction in migrating cells. (A) IKD-F11 Fas–cells cultured in doxycycline were transiently transfected with GFP-Xtfascin, PKC-mRFP and either wild-type Pak1-myc or Pak1AID-myc and allowed to migrate on 15 nM laminin for 6 hours. Cells were fixed and antibody-stained to detect Pak1 and imaged using FLIM to measure FRET. Pak1-AID inhibited the FRET interaction. Lifetime images are depicted with a pseudocolour scale where red is a low lifetime (1.85 nseconds) and blue is high (2.4 nseconds). Representative of at least eight cells in each sample. (B) Cumulative FRET efficiency graph for control cells or cells co-expressing the indicated Pak1 constructs. The mean FRET efficiency for each condition is from seven to nine cells per condition and three independent experiments. (C) Pak1 activity is Rac dependent. SW480-Pa cells were pretreated with BIM, NSC23766 or solvent (Con), as described in the Materials and Methods, plated on 15 nM laminin for 2 hours in the continued presence of inhibitors, then lysed and analysed by SDS-PAGE and immunoblotting for total Pak1 or Pak1–T423PO₄ as a reporter of Pak1 activation. Representative of three independent experiments.