Cells (1 × 106) expressing RUSH constructs per condition were treated with 40 μM biotin to induce trafficking of the reporter and incubated at 37°C. At the desired time point, cells were washed once with PBS supplemented with 0.5 mM EDTA and incubated with PBS supplemented with 0.5 mM EDTA for 5 min at 37°C. Plates containing cells were then put on ice. Cells were resuspended and transferred to ice-cold tubes for centrifugation at 300g for 5 min at 4°C. Cell pellets were resuspended in cold PBS supplemented with 1% FCS for blocking and incubated for at least 10 min. After centrifugation, cell pellets were incubated in a solution of Alexa Fluor 647–coupled anti-GFP (catalog no. 565197, BD Pharmingen) prepared in PBS supplemented with 1% serum for 40 min on ice. Cells were washed three times in cold PBS and 1% serum and fixed with 2% paraformaldehyde (Electron Microscopy Sciences) for 15 min. Cells were washed twice with PBS before acquisition with an Accuri C6 flow cytometer. The intensity of the GFP signal (FL1) and the Alexa Fluor 647–conjugated antibody (FL4) was measured on GFP-positive cells. The FL4 signal was divided by the FL1 signal to normalize for the transfection level. The FL4/FL1 ratio for each condition was then normalized to the DMSO control.

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