Wongthai P , Hagiwara K , Miyoshi Y , Wiriyasermkul P , Wei L , Ohgaki R , Kato I , Hamase K , Nagamori S , Kanai Y .

	Figure 1. Separation of p-boronophenylalanine (BPA) by HPLC. Chromatograms showing the separation of a BPA standard (0.2 pmol) (a), amino acid standards (0.2 pmol each) (b), a sample from the oocyte expressing LAT1 (c), and the control oocyte not expressing LAT1 (d). (a, b) The BPA peak was identified by retention time and spike study (not shown). Similarly, by comparison with amino acid standards and a spike study (not shown), the peaks neighboring BPA in oocyte samples were identified as alanine (Ala) and proline (Pro). (c, d) The LAT1-expressing oocyte and non-expressing control oocyte were incubated in the uptake buffer containing BPA. Samples from the oocytes were separated by HPLC. The increased BPA peak height in (c) showed that the uptake of BPA was mediated by LAT1. Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; His, histidine; Ser, serine.
	Figure 2. Chromatograms of p-boronophenylalanine (BPA) taken up by aromatic amino acid transporters. Chromatograms for oocytes expressing each transporter (solid line) and non-expressing control oocytes (dotted line) are overlaid on the same scaling. In ATB0,+ (b), LAT1 (c) and LAT2 (d), the BPA peaks were significantly higher than those in the controls, but not in the others. The endogenous alanine (Ala) and proline (Pro) peaks were frequently found to be lower in the oocytes expressing transporters, probably because of the translational consumption of endogenous amino acids.
	Figure 3. Time-dependent uptake of p-boronophenylalanine (BPA). Uptakes of BPA by oocytes expressing the indicated transporter (○) and by non-expressing controls (▾) were measured over 60 min in Na+ uptake buffer containing 50 μM BPA for ATB0,+ (a), and in Na+-free uptake buffer for LAT1 (b) and LAT2 (c). The time-course of the uptake was determined at 50 μM BPA because the measurement became less accurate for the 10- and 20-min time points at lower concentrations. The transporter-mediated uptakes (•) were determined by subtracting the uptake in non-expressing control oocytes (▾) from the uptake in oocytes expressing each transporter (○). Uptake by ATB0,+ and LAT2 linearly increased up to 30 min (a, c). Uptake by LAT1 was linear over 60 min (b). Each data point represents the mean ± SEM of three to five oocytes.
	Figure 4. Concentration-dependent uptake of p-boronophenylalanine (BPA). Uptake of BPA was measured for 30 min at 3–1000 μM for ATB0,+ (a), LAT1 (b) and LAT2 (c). The velocity (V) of transporter-mediated uptake was determined by subtracting the uptake in non-expressing control oocytes from the uptake in oocytes expressing each transporter. Representative Michaelis–Menten fitting (left panels) and Lineweaver–Burk plot (right panels) are shown for each transporter. Each data point represents the mean ± SEM of five to seven oocytes.
	Figure 5. Transporter expression and p-boronophenylalanine (BPA) uptake in cancer cell lines. (a) Expression level of LAT1 was analyzed by western blotting using crude membrane fraction. The cell lines were ordered in increasing LAT1 amounts. Na+/K+ ATPase was used as loading control. (b) Similarly, the expression of ATB0,+ was analyzed. ATB0,+ was expressed in MCF-7 cells, to a lesser degree in T3M4 cells, and not detected in the other cell lines. (c) Uptake of BPA in cell lines was measured for 5 min in the presence or absence of Na+. The presence of Na+ did not significantly affect the BPA uptakes in the cell lines. A representative result was shown with the mean ± SEM (n = 4).
	Figure 6. Differential roles of ATB0,+ and LAT1 in p-boronophenylalanine (BPA) uptake. (a) Uptake of BPA in MCF-7 cells was measured for 10 min at 100 μM (left) and 1000 μM (right) BPA. At 1000 μM, the uptake rate in the presence of Na+ increased by ∽1.4-fold from the Na+-free uptake. (b) MCF-7 cells were transfected with non-targeting control siRNA #1 and #2, and LAT1-targeting siRNA #3, #4 and #5. The siRNAs #3–#5 achieved a ∽80% reduction of LAT1 protein amount. (c) Uptake by ATB0,+ was separated by LAT1 knockdown and lysine inhibition. The uptakes in the presence of Na+ and with mock knockdown were set at 1.0 at each BPA concentration (bars 1 and 4). At 100 μM BPA, the uptake levels with LAT1 knockdown did not differ regardless of Na+ (bars 2 and 3). At 1000 μM, LAT1 knockdown left a significant Na+-dependent component (bars 5 and 6). Overall, although with various statistical significances, the BPA uptake in bar 5 was inhibited by 5 mM lysine to a level similar to bar 6 (bar 7). This Na+-dependent and lysine-inhibitable component accounted for at least 20–25% of the total uptake. siRNA#1 was used for control siRNA. n.s., not significant.

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