Figure 1

LAT1 is internalized and degraded in response to PMA. (A) Cells of the stable T-REx HeLa line expressing LAT1-GFP were incubated with 25 mM (NH₄)₂SO₄ for 18 h and then incubated in the presence of DMSO (control) or PMA (1 µM) for 4 h. The cells were fixed and the localization of LAT1-GFP was examined by confocal microscopy. (B) Quantification of PMA-induced LAT1-GFP internalization. The fluorescence signals of at least 60 cells from three independent experiments were quantified. The graph shows the percentage of intracellular immunofluorescence determined as detailed under Methods. Unpaired t-test, ***p < 0.001. Scale bars, 5 μm. (C) Cells of the stable T-REx HeLa line expressing LAT1-GFP were incubated with sulfo-NHS-biotin for 30 min at 4 °C. The cells were then incubated in the presence of DMSO (control) or PMA (1 µM final concentration) for the indicated times at either 4 °C, to block membrane trafficking (control reduction), or 37 °C. The cells were washed and the remaining surface biotin was then cleaved off except in a control sample (total surface). The biotinylated proteins were purified by affinity chromatography with streptavidin-coated beads. LAT1-GFP was then detected by immunoblotting with anti-GFP antibody. A blot representative of three independent experiments is shown. (D) Quantification of the immunodetected signals in three independent experiments performed as in C. Data are presented as percentages of total surface and error bars represent standard deviations. (E) Cells of the stable T-REx HeLa line expressing LAT1-GFP were incubated with DMSO (control) or 1 μM PMA for the indicated times. Cell extracts were immunoblotted with anti-LAT1 antibody, to detect endogenous LAT1 and LAT1-GFP, and with anti-actin antibody. (F) Quantification of the signals in immunoblots performed as in E. The graph shows the mean of three independent experiments. The error bars represent standard deviations.