Fig. 4

Effect of putative PKC phosphorylation sites on hLAT2 transport activity. a Five putative PKC phosphorylation sites in hLAT2 protein are shown. The putative PKC phosphorylation sites and the transmembrane regions in hLAT2 protein were predicted by the in silico analysis tools “ScanProsite” and “TMHMM-2.0”, respectively. b, c Alanine uptake activity and hLAT2 protein expression levels at cell membrane in a transient hLAT2 expression system were examined by a transport assay and Western blot analysis, respectively. S2 cells were transfected with WT hLAT2 (WT) or Triple mut hLAT2 (Triple mut) plasmid, and non-transfected S2 cells were used as a negative control (-). In transport assay, the cells were incubated with alanine (1 µM) in Na⁺-free HBSS buffer for 2 min at 37℃. Data are shown as the mean ± SD of values obtained from four (b) or three (c) separate experiments, each performed in duplicate. **, p < 0.01; NS, not significant. Western blot analysis was performed using protein samples 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. d The effect of PKC activation on WT and Triple mut hLAT2 transport activity was examined by a transport assay. Cells were prepared in the same way as explained in b, c, and the cells were treated with DMSO (0.1% (v/v), -) or PMA (1 µM) for 1 h. After removing the reagents, the cells were incubated with alanine (1 µM) in Na⁺-free HBSS buffer for 2 min at 37℃. Data are shown as the mean ± SD of values obtained from four separate experiments, each performed in duplicate. **, p < 0.01; NS, not significant