Movie S3.

Inactivation of RapR-Src disrupts Rac1 activation in HeLa cells. A HeLa cell coexpressing RapR-Src-as2-mCherry-myc and Rac1-FLARE (transient transfection) and iPEP-GFP(Y66S)-FRB (adenoviral infection) was imaged for 2 h every 2 min. Rapamycin (500 nM) was added after 30 min (indicated by appearance of “Rapamycin Added”) and 1NA-PP1 (250 nM) was added after 15 min (indicated by appearance of “1NA-PP1 Added”). The movie shows FRET/CFP signal ratio images of Rac1-FLARE biosensor in a living cell. Warmer colors reflect higher local activity of Rac1.