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The method of genotyping
The primer pairs used for the detection of Glu298Asp polymorphism of the heNOS gene,
resulting in the 457-bp fragment encompassing the missense Glu298Asp variant, were as
follows: sense 5'-TCC CTG AGG AGG GCA TGA GGC T-3' and antisense 5'-TGA GGG TCA
CAC AGG TTC CT-3'. Polymerase chain reaction (PCR) was carried out using a standard Taq
DNA polymerase kit (Takara Taq; Takara, Otsu, Japan). The amplification condition of 96C
denaturation for 1 minute, 60C annealing for 1 minute, and 72C extension for 1 minute was
used for 30 cycles in a thermal cycler (TC1; Perkin-Elmer, Foster City, CA).
The amplified products were digested by BanII (Takara) into small fragments (137 bp
and 320 bp) and separated by agarose gel (Figure 1A). It is noteworthy that a restriction site by
BanII is lost when there is a G to T substitution at the position of 1917 of the eNOS gene. G to T
conversion at nucleotide position 1917 of the eNOS gene (894 of eNOS cDNA) results in a
replacement of glutamic acid by aspartic acid at codon 298 (Glu298Asp). All fragments
uncleaved were double-checked by direct sequencing to avoid mistyping (Figure 1B).
The method of site directed mutagenesis
Briefly, the primer 5'-GCA GGC CCC AGA TGA TCC CCC AGA ACT CTT CC-3' was used to
create the heNOS (G1917T) mutant. G to T conversion at nucleotide position 1917 of the eNOS
gene resulted in a replacement of glutamic acid by aspartic acid at codon 298. Partial heNOS
plasmid was subcloned using the HincII (Takara) fragment (heNOS; 1396 bp) of the
pcDNA3.1-WT-heNOS plasmid into pBluescriptII (Stratagene, La Jolla, CA). First, PCR was
performed using the following combinations of primer pairs: 5'-GCA GGC CCC AGA TGA TCC
CCC AGA ACT CTT CC-3', M13 primer M4, and MUTB-4, M13 primer RV. Twenty-five cycles
of PCR were performed with denaturation at 95C for 30 seconds, annealing at 55C for 2
minutes, and extension at 72C for 2 minutes. Both PCR products in the amount of 0.5 L each
were put into the same tube together with 5 L 10x ExTaq buffer (Takara), 5 L 25 mM dNTP
mixture (Takara), and 36.5 L molecular grade water; and then hetero-duplex formation was
performed in 1 cycle of denaturation at 94C for 10 minutes, annealing of linear decrease of
temperature between 94C and 55C over 60 minutes, and extension at 37C for 15 minutes.
After adding 2.5 U of ExTaq, the product was heated on a thermal cycler at 72C for 3 minutes,
and both M13 primer M4 and M13 primer RV were added. Ten cycles of PCR were performed
with denaturation at 95C for 30 seconds, annealing at 55C for 1 minute, and extension at 72C
for 2 minutes. The second PCR product was digested with XbaI (Takara) and BamHI (Takara)
restriction endonucleases, and inserted into the pBluescriptII vector that had been digested with
the same enzyme. After confirming the mutagenesis of the heNOS fragment by DNA sequencing,
the fragment containing the codon 298 variant of heNOS was digested with NheI restriction
endonuclease and inserted into pcDNA3.1-WT-heNOS that had been digested with the same
enzyme.
The method of Western analysis
CHO cells (1x107) were dissolved in the ice-cold extraction buffer of the following composition:
1 mM dithiothreitol, 20 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate
(Sigma), 0.5 mM EDTA (pH 7.4), 0.5 mM EGTA (pH 7.4), and 50 mM Tris-HCl (pH 7.4).
Protease inhibitors phenylmethylsulfonyl fluoride (174 g/mL; Sigma), aprotinin (6 L/mL;
Sigma), and leupeptin (10 g/mL; Sigma) were added to the buffer and kept on ice. Samples
were homogenized 5 times on ice by Dounce homogenizer and centrifuged in 500 g for 10
minutes at 4 C. The supernatant was again centrifuged in 100,000 g for 20 minutes at 4 C.
After protein quantitation by Bradford assay reagent (Bio-Rad, Hercules, CA), protein
concentration of the lysates was adjusted to 50 g per lane in the sampling buffer (3.5% SDS,
100 mM DTT, 0.02% BPB, 20% glycerol, 60 mM Tris, pH 6.8). After 3 minutes' incubation in
boiling water, the lysates were electrophoretically separated on 7.5% polyacrylamide gel.
Western blotting was performed on nylon membranes (Hybond; Amersham, Buckingham, UK)
with monoclonal antibodies to heNOS (Transduction Lab, Lexington, KY). After the horseradish
peroxidase conjugated appropriate second antibody incubation, chemiluminescent signal
detection (ECL plus; Amersham) was performed using a cooled CCD camera system (Las-1000;
Fuji Film, Tokyo, Japan).