CVR-2010-1352R2
Detailed Methods
Measurement of Cellular Total NAD + /NADH Ratio
The bEnd.3 cells were treated with vehicle and 10 μmol/L βL
.
The treated cells were washed with ice-cold PBS, and harvested with 80% methanol. The cells were then lysed mechanically with a 31-gauge needle and centrifuged. The supernatant was transferred to a tube and the remaining pellet was lysed again by sonication in 80% methanol and centrifuged.
The supernatant was pooled in the same tube. All procedures were performed at a temperature of below 4°C. Electrospray-ionization mass spectrometry was performed in positive ion mode using a MDS Sciex API 4000 Triple Quadrupole Mass Spectrometer
(Applied Biosystems, Ontario, CA), followed by chromatographic separation with the Agilent
1100 series HPLC system (Agilent technologies, Palo Alto, CA) and XTerra MS C18 2.1 x
150 mm with a 3.5 μm column (Waters, Milford, Massachusetts, USA) as described previously.
1
Determination of pyruvate/lactate ratio
The bEnd.3 cells were treated with vehicle and 10 μmol/L β-lap for 15 minutes. The treated cells were washed with ice-cold phosphate buffered saline, and quickly deproteinized in 400 μl of acid extraction buffer (1 mol/L HClO
3
). The cells were then lysed by repeated sonication and centrifuged at 13,000 rpm for 10 minutes. Following centrifugation, the supernatant was transferred to a tube and neutralized with 500 μl of 1 M/L borate buffer (pH 11). The pyruvate/lactate ratio representing cytosolic free NAD
+
/NADH ratio
2
was measured using a
D-lactate assay kit (Megazyme International, Bray, Ireland), according to the manufacturer's instructions.

CVR-2010-1352R2
Measurement of eNOS Activity
The conversion of [
14
C]L-arginine to [
14
C]L-citrulline was used to assess eNOS activity in cultured bEND.3 cells using the NOS Activity Assay Kit (Cayman Chemicals, Inc., Ann Arbor,
MI, USA). Briefly, bEnd.3 cells, grown in 150 mm culture dishes, were treated with β-lap vehicle (DMSO) or βL (10 μmol/L) for 5 or 15 minutes. Cells were washed with PBS and harvested in PBS containing 1 mmol/L ethylenediamine tetraacetic acid (EDTA). To prepare total cell lysates, cells were centrifuged (5,000 g, 2 minutes), and the cell pellet was sonicated briefly in homogenization buffer (25 mmol/L Tris∙HCl, pH 7.4, 1 mmol/L EDTA, and 1 mol/L
EGTA) to disrupt the cells. The cell homogenate was centrifuged (16,000 g, 15 minutes, 4°C).
The supernatant containing eNOS was separated, and samples were incubated with a reaction solution (50 mmol/L Tris∙HCl, pH 7.4, 6 µmol/L tetrahydrobiopterin, 2 µmol/L flavine adenine dinucleotide, 2 µmol/L flavin mononucleotide, 1.25 mmol/L NADPH, and 0.75 µmol/L CaCl
2
) and 1 µCi [ 14
C]L-arginine at 30°C for 60 minutes. The reaction was stopped by the addition of
400 µL of cold stop buffer (50 mmol/L HEPES, pH 5.5, 5 mmol/L EDTA). Equilibrated resin
(100 µL) was added to each reaction sample. Following centrifugation, radioactivity in the eluate was quantified. Radiolabeled counts per minute of [
14
C]L-citrulline generation were measured and used to determine NOS activity.
Measurement of [Ca 2+ ]i
The [Ca 2+ ] i
in cells was measured using the fluorescent dye fluo-4/AM (Invitrogen,
Carlsbad, CA). bEnd.3 cells adherent to μ-Slide (ibidi GmbH, Munich, Germany) were washed three times with PBS (pH 7.4) and incubated with complete DMEM (10% FBS) containing fluo-4/AM for 1 hour at 37°C, 5% CO
2
. During incubating with fluo-4/AM, the cells were also treated with BABTA-AM (20 μmol/L), 8-Br-cADPR (100 μmol/L), ES936 (5
μmol/L) or dicoumarol (5 μmol/L) for 45 minutes, or EGTA (2 mmol/L) for 2 minutes. The

CVR-2010-1352R2 cells were then washed three times with PBS and scanned with the confocal microscopy system (LSM 510 META; Carl Zeiss, Jena, Germany). During scanning, 20 μmol/L of βL was treated and green fluorescence was detected for every second until 5 minutes.
Quantification of relative fluorescence increase was measured using the regions of interest
(ROI) tool.
Western Blot Analysis
The samples were washed with PBS and homogenized in an ice-cold lysis buffer containing
0.1 mmol/L sodium vanadate, 1 mmol/L phenylmethanesulphonylfluoride, 25 mmol/L NaF, 50 mmol/L Tris-HCl, 40 mmol/L β glycol phosphate, 120 mmol/L NaCl, 1% NP40, and 0.5%
Triton X 100. The cells were washed with ice-cold PBS and lysed with ice-cold lysis buffer.
Centrifugation (12,000 g , 10 minutes) was performed three times, and the soluble protein was then determined using the Bradford assay. Protein abundance and phosphorylation in isolated protein were analyzed using western blot according to standard procedures.

CVR-2010-1352R2
References
1. Lazzarino G, Amorini AM, Fazzina G, Vagnozzi R, Signoretti S, Donzelli S et al.
Single-sample preparation for simultaneous cellular redox and energy state determination.
Anal Biochem 2003; 322 :51-59.
2. Williamson DH, Lund P, Krebs HA. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J 1967; 103 :514-527.