*4.8. Immunofluorescence*

LAT1 and LAT2 expression were examined by immunofluorescence in JEG-3 cells transfected with scramble or raptor siRNA. The cells were fixed with methanol at −20 ◦C for 5 min. After permeablilized by Triton X-100, cells were blocked in goat serum and then incubated with anti-LAT1 or anti-LAT2 antibody overnight at 4 ◦C. After several washes, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG for 1 h at 37 ◦C, and then with dye 4,6-diamido-2-phenylindole (DAPI) for 15 min to counterstain nuclei. After three washes in PBS, coverslips were mounted in a drop of 30% glycerol. Images were acquired with a NikonE800 fluorescence microscope.
