Supplemental procedure
Construction of plasmid p3XFLAG-CMV14-hASCT2
Total RNA of HeLa S3 cells was extracted by Isogen II (Nippon Gene; Japan)
following the manufacturer’s instruction. First strand cDNA of HeLa S3 cells was
synthesized from total RNA by using SuperScript III first-strand synthesis system with
oligo dT primer (Invitrogen; CA, USA). Coding sequences of ASCT2 (CCDS12692.1) was
amplified
from
HeLa
S3
cDNA
template
CTCTTTCAGGGACCCATGGTGGCCGATCCTCCT-3’
using
and
ACCGCGTGGCACCAGCATGACTGATTCCTTCTCAGAG-3’.
forward
primer
5’-
reverse
primer
5’-
reaction
was
PCR
conducted by using KOD FX DNA polymerase (Toyobo; Japan). cDNA of ASCT2 was
cloned into pTriEx-6 vector (Novagen; Germany) by In-fusion kit (Clontech; Mountain
View) to obtained pTriEx6-hASCT2. The ASCT2 from pTriEx6-hASCT2 was then
amplified by using forward primer 5’-ATAAAGCTTATGGTGGCCGATCC-3’ and reverse
primer 5’-CCCTCTAGACATGACTGATTCCTTC-3’. PCR reaction was conducted by
using PrimeSTAR Max DNA polymerase (Takara; Japan). The ASCT2 was subcloned into
p3XFLAG-CMV-14 (Sigma-Aldrich; MO, USA) at HindIII and XbaI restriction sites to
obtain p3XFLAG-CMV14-hASCT2.
DNA transfection, membrane extraction, western blot and ASCT2 detection
HEK 293T (ATCC) was cultured in Dulbecco’s Modified Eagle Medium (Wako;
Japan) with supplement of 10% fetal bovine serum (Gibco; NY, USA) at 37oC, 5% CO2 and
humidity. The cells were transfected with p3XFLAG-CMV14-hASCT2 or p3XFLAGCMV14 (for mock cells) by using Lipofectamine LTX with Plus reagent (Invitrogen; CA,
USA) and further cultured for 48 hours. Transfected cells were collected and the crude
membrane fraction was obtained as described (Khunweeraphong et al).
Equal amount of the membrane fraction from the cells transfected with ASCT2 and
mock cells were dissolved in suspension buffer containing 1% Fos-Choline 12 (Affymetrix;
CA, USA) and incubate on ice for 10 min. Membrane samples were then mixed with the
Laemmli sample buffer containing 100 mM DTT and subjected to SDS-PAGE. After SDSPAGE, separated proteins were transferred electrophoretically to Hybond P PVDF
membrane (GE Healthcare Life Science; PA, USA). The membrane was blocked for 1 h at
room temperature with TBS-T (10 mM Tris pH 7.6 + 150 mM NaCl + 0.1% Tween 20)
containing 5% (w/v) skim milk. The membrane was then incubated for 2 h at room
temperature with TBS-T containing 5% (w/v) skim milk and 1:5,000 dilution of the primary
antibodies; either anti-ASCT2 polyclonal antibody produced in rabbit or anti-FLAG
polyclonal antibody produced in rabbit (Sigma-Aldrich; MO, USA). The membrane was
washed with TBS-T and subsequently incubated for 1 h at room temperature with TBS-T
containing 5% (w/v) skim milk and 1:5,000 dilution of horseradish-peroxydase-conjugated
anti rabbit IgG (Jackson ImmunoResearch Laboratory; PA, USA). The membrane was
washed again with TBS-T. The signals on membrane were developed by SuperSignal West
Femto Maximum Sensitivity Substrate (Thermo Scientific; IL, USA) and visualized under
the LAS-4000 mini Luminescent image analyzer (Fujifilm; Japan).
Reference:
Khunweeraphong N, Nagamori S, Wiriyasermkul P, Nishinaka Y, Wongthai P, Ohgaki R,
Tanaka H, Tominaga H, Sakurai H, Kanai Y.
J Pharmacol Sci. 2012;119(4):368-80. Epub 2012 Jul 31.
Figure legend for supplement Figure 1
Western blots were performed on HEK 293Tcells transfected human 3FLAGASCT2 as described in Supplemental procedure. In the western blots, the polyclonal antiASCT2 antibody recognized a band that is also detected with anti-FLAG antibody for the
transfected cells, confirming the specificity of the anti-ASCT2 antibody used in this study.