Gene Ensembl Gene ID Primer sequence Amplicon size(BP)

DDAH1 ENSMUSG00000028194 Forward (5'to 3') CCCAGCAAAGGGCATGTC

Reverse (5' to 3') CCATCTCCGAGTTGCTCACA

124

DDAH2 ENSMUSG00000007039 Forward (5'to 3') CCTGGTGCCACACCTTTCC

Reverse (5' to 3') AGGGTGACATCAGAGAGCTTCTG

88

Alpha Tubulin 2 ENSMUST00000077577 Forward (5'to 3') GCCTTCTAACCCGTTGCTATCA

Reverse (5' to 3') CGGTGCGAACTTCATCGAT

264

Supplementary Table S1.

Primer sequences used for PCR amplification of DDAH1, DDAH2 and tubulin message

s1A s1B

s1C

Supplementary Figures

s1D

Supplementary Figure S1. Conscious cardiovascular hemodynamics and activity during 24 hour period (shaded area represents 12 hour dark cycle) in naive DDAH1+/- (white squares) and DDAH1+/+ (black triangles). A) systolic blood pressure; p=ns B) Diastolic blood pressure; p=ns C) heart rate; p=ns D) pulse pressure; p=ns.

1E*

Supplementary Figure S1e Conscious cardiovascular hemodynamics and activity during 24 hour period (shaded area represents 12 hour dark cycle) in naive DDAH1+/- (white squares) and DDAH1+/+ (black triangles). E) Activity *p<0.05 Two Way ANOVA as indicated. n≥ 6.

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S1f Representative superimposed blood pressure traces from DDAH1+/- (red) and DDAH1+/+ (blue) anaesthetised mice after 24 hours LPS treatment and subsequent vasopressor (phenylephrine) challenge as indicated with arrow. DDAH1+/- mice show an enhanced and more prolonged responsiveness to vasopressor challenge compared to DDAH1 +/+ mice where blood pressure decays to baseline within 12-13 minutes

DDAH1 4 LGHPA-AFGRATHAVVRALPESLGQHALRSAKGEEVDVARAERQHQLYVGVLGSKLGLQV 62 +G P GR +HA++R +PESL A +D+A+A+R+H + G L +LGLQ+ DDAH2 1 MGTPGEGLGRCSHALIRGVPESLASGEGAGAGLPALDLAKAQREHGVLGGKLRQRLGLQL 60 DDAH1 63 VELPADESLPDCVFVEDVAVVCEETALITRPGAPSRRKEVDMMKEALEKLQLNIVEMKDE 122 +ELP +ESLP + D AV+ +TALITRP +P+RR EVD +++AL+ L L IVE+ DE DDAH2 61 LELPPEESLPLGPLLGDTAVIQGDTALITRPWSPARRPEVDGVRKALQDLGLRIVEIGDE 120 DDAH1 123 NATLDGGDVLFTGREFFVGLSKRTNQRGAEILADTFKDYAVSTVPVADGLHLKSFCSMAG 182 NATLDG DVLFTGREFFVGLSK TN RGAEI+ADTF+D+AVSTVPV+ HL+ C M G DDAH2 121 NATLDGTDVLFTGREFFVGLSKWTNHRGAEIVADTFRDFAVSTVPVSGPSHLRGLCGMGG 180 DDAH1 183 PNLIAIGSSESAQKALKIMQQMSDHRYDKLTVPDDIAANCIYL--NIPNKGHVLLH 236 P + GSS++AQKA++ M ++DH Y LT+PDD AA+C++L +P LLH DDAH2 181 PRTVVAGSSDAAQKAVRAMAVLTDHPYASLTLPDDAAADCLFLRPGLPGVPPFLLH 236

S2 Alignment of human DDAH1 and DDAH2 amino acid sequences. Red boxes indicate highly conserved region close to L-257 binding pocket. Highlighted residues indicate single residue difference between the two proteins: Glycine 129 (G129) in DDAH1 which is a threonine equivalent in DDAH2.

*

Figure S3 Cytokine profile in primary hepatocytes obtained from DDAH1+/+ (black bars) and DDAH1 -/- (grey bars) mice or from WT mice treated ex vivo with L-257 100 mM A) IFN gamma; B) IL1 beta C) IL-6 D) TNF alpha. n=6 *p<0.05 One Way ANOVA vs. DDAH1 +/+ as indicated.

s3A s3B

s3C s3D