Methods
Electrophoretic Mobility Shift Assay.
The sequence of the double-stranded nuclear factor (NF)-[kappa]B oligonucleotide was sense 5'-AGT TGA GGG GAC TTT CCC AGG C-3'; antisense 3'-TCA ACT CCC CTG AAA GGG TCC G-5' (Promega, Madison, WI). The oligonucleotides were 3' biotin end-labeled (Biotin end-labeling kit, Pierce, Rockford, IL), and whole aorta protein extracts were submitted to electrophoretic mobility shift assay (EMSA) by using a nonradioactive chemiluminescent EMSA kit according to the recommendations of the manufacturer (Lightshift chemiluminescent EMSA kit, Pierce). Epstein-Barr nuclear antigen extract served as internal control. Band intensities were measured by means of a reflectance scanner and Quantity One Quantitation Software (Bio-Rad). Experiments were performed in duplicate.
Table 1 online supplement : Primers used in the quantitative real-time PCR studies
Gene / NCBI GenBank / Sense (5'-3') / Antisense (5'-3') / Amplicon lengthaccession number / (pb)
Kir 6.1 / D42145 / Kcnj8 / GAAGAGTGGCCTGGAGTCTG / CGCAGAAGTGAATGACCTGA / 58
Kir 6.2 / NM_031358 / Kcnj11 / CCTCCTATCTGGCTGACGAG / GTGGGCACTTTAACGGTGTT / 118
SUR2A / D83598 / CGGGCCTTGTTTTAGTCTTTTCTGA / TAGAAAAGAGGCCATTCTTGTGCTG / 90
SUR2B / AF087838 / GTATGCCTGGGAGCACATTT / GGTTTCAGGTTGTTGCCACT / 183
BK alpha / AF083341 / Kcnma1 / TCAGCAGATGCATGTTTGAT / AAGAGATTGGCAAGCATTGT / 281
iNOS / NM_012611 / AGGGAGTGTTGTTCCAGGTG / TCTGCAGGATGTCTTGAACG / 81
β-actine / NM_031144 / CCTCTATGCCAACACAGTGCTGTCT / GCTCAGGAGGAGCAATGATCTTGA / 128
Table 2 online supplement: antibodies used in the Western blot experiments
Primary antibody / Dilution factor / Secondary antibody/HRP / Dilution factorAnti-SUR2A (BNJ-39) rabbit
Anti-SUR2B (BNJ-40) rabbit / 1/2 000 / Polyclonal donkey anti-rabbit IgG (GE Healthcare) / 1/10 000
Anti-BK alpha mouse
(BD Biosciences) / 1/500 / Polyclonal sheep anti-mouse IgG (GE Healthcare) / 1/1000
Anti-Kir6.1 chicken / 1/10 000 (vessel)
1/1 000 (heart) / Rabbit anti-chicken IgY
(Millipore) / 1/1 000
Anti-Kir6.2 goat IgG polyclonal
(Santa Cruz Biotechnology, Inc.) / 1/500 / Polyclonal donkey anti-goat
(Santa Cruz Biotechnology, Inc.) / 1/5 000
Anti-α-tubulin mouse IgG1 monoclonal, clone B-5-1-2
(Sigma-Aldrich) / 1/4 000 / Polyclonal sheep anti-mouse IgG
(GE Healthcare) / 1/4 000
The Kir6.1 primary antibody was kindly provided by William A. Coetzee (NYU School of Medicine, New York, USA)
and the Kir 6.2 by NQ Shi (Department of Medicine, University of Wisconsin, Madison, USA).
Figure 1 online supplement. Histogram plots representing the evolution of MAP (panel A) and aortic blood flow (panel B) in CLP6h and CLP18h groups with or without a one-hour infusion of PNU-37883A infusion (1.5 mg/kg bolus followed by 1 mg/kg/h). For each group, the column represents the final value compared to baseline after a 1h observation period.
Values are mean ± SD (n = 7 per group)
* p < 0.05 versus PNU-37883A group
Figure 2 online supplement.
Histogram plots representing maximal aortic blood flow (ABF) obtained after administration of a bolus of 1 µg/kg of norepinephrine in rats treated or not with PNU-37883A.
CLP6h and CLP18h: peritonitis observed after 6 hours and 18 hours; CLP6h or CLP18h + PNU: peritonitis observed after 6 or 18h and treated with a one-hour infusion of PNU-37883A (1.5 mg/kg bolus followed by 1 mg/kg/h). Values are mean ± SD (n = 7 per group)
* P < 0.05 vs. basal values; ** P < 0.05 vs. other groups
Figure 3 online supplement. Western blots revealing BKα subunit expression in aorta lysates in the various studied groups. Plots show the densitometric intensities for each condition, normalized against tubulin expression and presented in arbitrary units.
CLP6h, CLP18h and CLP30h: peritonitis observed after 6,18 and 30 hours; CLP6h or CLP18h + 1400W: peritonitis observed after 6h and 18 hours and treated intraperitoneally with 20 mg/kg of the selective iNOS inhibitor1400W
Values are mean ± SD (n = 7 per group)
Figure 4 online supplement. Aortic (A) and heart (B) iNOS mRNA expression normalized to β-actin
CLP6h and CLP18h: peritonitis observed after 6 and 18 hours; CLP6h + AG: peritonitis observed after 6h and treated intraperitoneally with 100 mg/kg of aminoguanidine (AG). Values are mean ± SD (n = 7 per group)
* p < 0.05 vs. sham, ** p < 0.05 vs. other groups.
Figure 5 online supplement: Quantification of the amplitude of NO-Fe(DETC)2 in aorta (A) (n= 7) and quantification of the amplitude of O2-CMH signal in aorta (n=7) (B)
CLP6h and CLP18h: peritonitis observed after 6 and 18 hours; CLP6h + 1400W: peritonitis observed after 6h and treated intraperitoneally with 20 mg/kg of 1400W.
p< 0.05 vs. sham; ** p< 0.05 vs. other groups