Confocal laser-scanning microscopy was conducted as described previously [35]. Post 48 h of plasmid transfection, cells were fixed with ice-cold methanol. After washing with PBS (–), the cells were treated with TO-PRO-3 iodide (Molecular Probes, Eugene, OR, USA) to visualize nuclei. After a subsequent wash with PBS (–), the cells were mounted on Fluorescence Mounting Medium (Agilent, Santa Clara, CA, USA). Fluorescence was detected using the FV10i Confocal Laser Scanning Microscope (Olympus, Tokyo, Japan) to analyze the localization of EGFP-fused transporter proteins.
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