For measurement of microtubule stability in live cells, U2OS cells were transfected with YFP-EML4-WT or S144/146A for 24 hours before being grown in microwell 8-well chamber slides (Ibidi). Cells were incubated with 25 nM SiR-Tubulin (Cytoskeleton Inc.) for 4 hours, and MG132 was added to the media 30 min before imaging. Fields of view containing transfected cells with bipolar mitotic spindles were selected, and media were supplemented with nocodazole (200 μg/ml) along with MG132 and SiR-Tubulin, and image capture immediately commenced. Z stacks comprising sections of 29 μm by 0.5 μm were captured every 60 s for 15 min using a VisiTech VT-Infinity3 confocal microscope fitted with a Hamamatsu C11449-22CU Flash 4.0 V2 sCMOS camera and Nikon Plan Apo 100× objective (numerical aperture, 1.47). Images were cropped to single cells and deconvolved before analysis for SiR-Tubulin intensity in MATLAB.

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