Figure 5

PMA-induced ubiquitylation of LAT1^{K19R,K25R,K30R} is impaired. (A) Cells of the stable T-REx HeLa lines expressing LAT1-GFP or LAT1^{K19R,K25R,K30R}-GFP (hereafter noted as LAT1(3KR)-GFP) were incubated with 25 mM (NH₄)₂SO₄ for 18 h and 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. Scale bars, 6 μm. (B) Quantification of the LAT1-GFP internalization shown in C. The images of at least 35 cells from three independent experiments were examined. The graph shows the percentage of intracellular immunofluorescence measured for each cell, determined as detailed in Methods. Unpaired t-test, ***p < 0.001. (C) Cells of the stable T-REx HeLa line expressing LAT1-GFP or LAT1(3KR)-GFP were incubated with DMSO (control) or 1 μM PMA for the indicated times. Cell extracts were immunoblotted with anti-GFP and anti-actin antibodies. (D) Quantification of the signals in immunoblots performed as in C. The graph shows the mean of three independent experiments. The error bars represent standard deviations. (E) Cells of the stable T-REx HeLa lines expressing LAT1-GFP or LAT1(3KR)-GFP were incubated with DMSO (control) or 1 μM PMA for 30 min. LAT1-GFP immunoprecipitates were prepared and probed with anti-Ub and anti-GFP antibodies. Dot indicates an unspecific band. (F) Quantification of the ubiquitylated LAT1-GFP signals immunodected in three independent experiments performed as in E. Data are presented as percentages with respect to the untreated control and error bars represent standard deviations. Unpaired t-test, *p < 0.05 (PMA-treated cells expressing LAT1-GFP compared to PMA-treated cells expressing LAT1(3KR)-GFP).