Endocytosis assay

Exponentially growing cells were induced for 4 hours in 2% galactose to allow for the production of GFP and Htt-GFP proteins. Cells were washed in 5 ml cold YP, re-suspended in 250 µl cold YP containing 20 µg/ml FM4-64 (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide; Life Technologies, Carlsbad, CA), and stained in darkness for 30 minutes in ice/water slurry. After being re-suspended in 5 ml cold YP a 1 ml sample, corresponding to time point zero, was washed one more time in cold YP and stored on ice. The remaining cells were spun down and re-suspended in 4 ml pre-warmed YP containing 2% galactose. Endocytosis was initiated by incubating the cells at 30 °C followed by a 30 min chase. Samples (1 ml) were washed twice in cold YP and stored on ice until analysis. Z-stacks were imaged for visible FM4-64 stained vesicle(s).