Rho activity does not modulate the interaction of fascin-1 with conventional protein kinase C (cPKC). (A) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCα- monomeric red fluorescent protein (mRFP) in C2C12 cells on fibronectin (FN). (B) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCγ-mRFP in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C), Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM.*P < 0.01 versus control.