Cells were transfected with GFP-EB1ΔC-2xEGFP encoding plasmid using FuGENE 6 Transfection Reagents (Promega Corporation), according to the manufacturer’s instructions. The construct was a generous gift from Dr. T. Wittmann (University of California, San Francisco, CA) (20 (link)). The cells with physiologic levels of GFP-EB1 expression were imaged 48 hours post-transfection using Yokagawa CSU-X1 spinning disk confocal microscope under a 100× objective (Zeiss). For each cell, hundreds of GFP-EB1–labeled individual microtubule tips were imaged. Images were acquired every second for 3 minutes for a total of 180 time-lapse images per cell; approximately 10–20 representative cells per condition. Image analysis was performed using the MATLAB-based plusTipTracker algorithm, as described previously (21 (link)). The following parameters were used: search radius 5–10 pixels, max gap length 12, max shrinkage 1.0, max angle forward 25, max angle backward 10, and fluctuation radius 2. All parameters were calculated per cell; for each cell line, 10 videos containing one or two cells were acquired. Mann–Whitney test was used to calculate any statistical difference in the mean parameter values.