2 amino 2 norbornanecarboxylic acid bch

Glutamine was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Glutamic acid dimethylester hydrochloride (dm‐Glu) was purchased from Tokyo Chemical Industry (Tokyo, Japan). [U‐¹³C]‐Glucose, [U‐¹³C]‐Glutamine and 2‐amino‐2‐norbornane carboxylic acid (BCH) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). CB‐839 was from Selleck chemicals (Houston, TX, USA). Small interfering RNAs for mouse Gls and Gls2 were purchased from Horizon Discovery (Cambridge, UK). Fura‐2 acetoxymethyl ester (Fura‐2 AM) was from Dojindo (Kumamoto, Japan). Anti‐GLUD1/2 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti‐actin antibody was from Calbiochem, Merck KGaA (Darmstadt, Germany).

For the synthesis of 3‐fluoro‐l‐α‐methyl[carboxyl‐¹⁴C]tyrosine ([¹⁴C]FAMT), 3‐fluoro‐4‐methoxyphenylacetone as a starting material was purchased from NARD Institute (Amagasaki, Japan). [¹⁴C]FAMT was synthesized by Sekisui Medical (Tokyo, Japan) to obtain high specific radioactivity by Bücherer–Strecker reaction.26 [¹⁴C]FAMT was identified by the analysis of ¹H‐nuclear magnetic resonance (AV400M; Bruker Biospin, Rheinstetten, Germany), high performance liquid chromatograph (Agilent 1200) and mass spectrum (LTQXL; Thermo Fisher Scientific, Waltham, MA, USA).25 The purity of the [¹⁴C]FAMT determined on high‐performance liquid chromatography was 99% and its specific radioactivity was 1.77 GBq/mmol.
l‐[¹⁴C]Leucine and l‐[¹⁴C]Alanine were purchased from Moravek Biochemicals (Brea, CA, USA). l‐[¹⁴C]Methionine and l‐[¹⁴C]Cystine were from American Radiolabeled Chemicals (St. Louis, MO, USA). l‐[¹⁴C]Glutamine (10.1 GBq/mmol) and l‐[¹⁴C]Tyrosine were from PerkinElmer (Boston, MA, USA) and Amersham Biosciences (Buckinghamshire, UK), respectively.
Non‐radiolabeled FAMT was purchased from NARD Institute (Amagasaki, Japan).25 Amino acids and 2‐amino‐2‐norbornanecarboxylic acid (BCH) were from Sigma‐Aldrich (St. Louis, MO, USA). Other general chemicals were from Wako (Osaka, Japan).

To confirm the uptake ability of ²¹¹At‐AAMT by PANC‐1 cells via LAT1, cells were seeded into 24‐well plates (1 × 10⁵ cells/mL) and cultured for 2 d. Following incubation in Hanks’ balanced salt solution for 30 min, cells were treated with ²¹¹At‐AAMT and other reagents. ²¹¹At‐AAMT was added 1 kBq to each well. After treatment, cells were washed twice with PBS(−), lysed with 0.1 N NaOH and radioactivity was measured with a gamma counter 2480 Wizard² (PerkinElmer, Inc). Protein levels were measured on a plate reader using a BCA protein assay kit (FUJIFILM Wako Pure Chemical Corporation). We used 2‐amino‐2‐norbornanecarboxylic acid (BCH, Sigma‐Aldrich) as the LAT inhibitor. Uptake was measured after 0.5, 1, 2, 5, 10, and 30 min. The uptake assay was also performed using 3 cell lines: Mock‐HEK293, hLAT1‐HEK293, and hLAT2‐HEK293. The procedure implemented was the same as that used for the cancer cell lines. The incubation time was 10 min. BCH was used at 200 mmol/L and AMT at 1 mmol/L. Mock treatment was represented by cells expressing an empty vector. We evaluated LATs affinity by the uptake inhibitory effects of ²¹¹At‐AAMT for LAT1 and LAT2 in the presence or absence of BCH, and non‐labeled AMT.¹⁵, ²⁶