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tra12161-sup-0001-FigureS1.jpgJPEG image609KFigure S1: Monitoring the trafficking of FLAG-furin from the plasma membrane to the Golgi apparatus in cell populations by FACS. HeLa cells were transfected with FLAG-furin for 24 h. Monolayers were harvested with 5 mm EDTA to obtain a cell suspension and incubated with rabbit anti-FLAG antibodies for 45 min on ice, washed in PBS and either fixed (0 min) or incubated in serum-free media for up to 90 min at 37°C to internalize the antibody-FLAG-furin complexes. Immediately following the incubation, cell suspensions were fixed and permeabilized and stained with Alexa568-conjugated anti-rabbit IgG (red). GM130 was stained with mouse monoclonal anti-GM130 antibodies, followed by Alexa488-conjugated anti-mouse IgG (green). A) Shown are 3D reconstructed confocal images presented as 2D projections. Bar represents 10 µm. B) Histograms of pulse-width values for FLAG-furin and GM130 are shown to provide a complete view of the dataset (n = 2) for each time-point. Thickness of line represents SEM of the dataset at each mean value.

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