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Fig. 5 | The Journal of Physiological Sciences

Fig. 5

From: Protein kinase C activation upregulates human L-type amino acid transporter 2 function

Fig. 5

Effect of PKC activation on hLAT2 localization at cell membrane. a The effect of PKC activation on the transport kinetics of hLAT2-mediated alanine uptake was examined by a kinetic analysis. S2-Mock cells and S2-LAT2 cells were treated with DMSO (0.1% (v/v)) or PMA (1 µM) for 30 min. Pre-treated cells were incubated with alanine (1, 100, 200, 400, 1000 and 2000 µM) in Na+-free HBSS buffer for 1 min at 37℃. Transport activity levels were calculated by subtracting the value obtained from S2-Mock cells from the value obtained from S2-LAT2 cells. Kinetic parameters were estimated by carrying out Michaelis–Menten-type non-linear curve fitting. Data are shown as the mean ± SD of values obtained from three separate experiments, each performed in duplicate. *, p < 0.05; **, p < 0.01 vs DMSO; NS, not significant. b The effect of PKC activation on hLAT2 protein localization was examined by confocal microscopy analysis. S2 cells transfected with WT hLAT2 plasmid were treated with DMSO (0.1% (v/v)) or PMA (1 µM) for 1 h. After removing the reagents, cells were fixed with 4% paraformaldehyde and the localization of EGFP-tagged WT hLAT2 protein (green) was examined by confocal microscopy. DAPI (cyan) was used for nuclear counter-staining. Experiments were repeated three times and representative images are shown. Scale bar, 10 µm. c The effect of PKC activation on hLAT2 protein expression levels at cell membrane was examined by Western blot analysis. S2-LAT2 cells were treated with DMSO (0.1% (v/v)) or PMA (1 µM) for 30 min, and protein samples were prepared with ultracentrifugation method. β-Actin and Na+/K+-ATPase α1 were both used as loading controls and also used as a cytosol marker and a cell membrane marker, respectively. Representative results that were obtained from three independent experiments are shown

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