A, The cells were transfected with ECFP-OTK18-EYFP 24 hours prior to imaging, and treated with or without 20 µg/ml puromycin at t = -30 min, then with or without 10µM MG132 t = 0 min (the start time of imaging). Images were taken in 5-minute increments up to 120 minutes. Cells were excited with CFP, and both CFP and YFP emissions are shown as well as the calculated SE-FRET image. 3D surface plots were constructed using ImageJ. B, Average nF intensity was calculated from time-lapse images using MetaMorph software and plotted as fold increase over original average nF at t=0 in the absence of puromycin (left panel) or presence of 20 mg/ml puromycin (right panel). Solid triangle (black) or square dots (green) represent MG132 untreated or treated groups, respectively. Linear regression and repeated measurement ANOVA (RMANOVA) were performed by Prism (p > 0.05 for no puromycin-treated experiment, and p < 0.0001 for puromycin-treated experiment).