A Low salt diet. C Low salt diet + mf4-31c1 3. D High salt diet + mf4-31c1 3. B High salt diet
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2 A Low salt diet GV [AU]:. [mmhg]: B High salt diet GV [AU]:. [mmhg]: C Low salt diet + mf-c GV [AU]:. [mmhg]: D High salt diet + mf-c E Lymph capillary density (AU) GV [AU]: 9. [mmhg]: # LSD HSD * * 0 0 # Control mf-c Control mf-c 0 (mmhg) Supplemental Figure : Panels (A-D) Whole mount stainings of lymph capillaries (anti-lyve- antibody) in ears of FVB mice fed LSD or HSD, with and without mf-c treatment. Red square is the computerized quantitated area; numerical value is lymph-capillary density (arbitrary units) which is given together with mean arterial blood pressure (; mmhg) for each individual animal. (E) Lymph-capillary density in ear and mean arterial blood pressure () in the mice.
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5 VEGFR Prox- Anti-VEGFR C E B D F WT A K-FLT Supplemental Figure : Lymph capillaries in kidneys in wild type mice (WT) and in mice with expression of soluble VEGFR under the control of the keratinocyte receptor (K-FLT mice). Green: VEGFR reporter fluorescence in wt and K FLT mice (Panels A and B); red: Prox- reporter fluorescence expression in WT and in K-FLT (Panels C and D). Panels E and F: anti-vegfr staining (brown) of renal lymph vessels in the same mice. In contrast to the hypoplastic lymph vessels in the skin, K-FLT mice showed normal lymph vessels in the kidney.
6 Supplemental Table : Differences in Na + concentration and osmolality between plasma and microdialysate from skin interstitium in rats. LSD: low salt diet, HSD: high salt diet. LSD (n=0) HSD (n=0) a) Na + concentration (mmol/l) Plasma 0.8±8. 8.±. Microdialysate 9.±8. 9.±9.0 P value (LSD versus HSD) plasma: 0.; microdialysate: 0.9 b) Osmolality (mosmol/kg) Plasma 09.±7.9 0.±0. Microdialysate.±.7.7±8.8 P value (LSD versus HSD) plasma: 0.; microdialysate: 0.7 P value (plasma versus microdialysate) LSD: 0.0 HSD: <0.00 LSD: 0. HSD: 0.0
7 A WT -probe 9. kb Long Arm of Homology (. kb) 9. kb loxp_fo loxp_re Short Arm (. kb) int. probe Target Region ( bp) Targeting construct Neo recombined -probe.7 kb loxp_fo Neo. kb int. probe loxp_re B C appr. kb loxp FRT Supplemental Figure. Generation of TonEBP-floxed mice. Panel A. Targeting Strategy. TonEBP-floxed mice were generated by ingenious Targeting Laboratory, Inc. (00 Smithtown Avenue, Ronkonkoma, NY 779) using standard gene-targeting techniques. The targeting construct was designed such that the short homology arm extends about. kb to exon, whereas the long homology arm extends about. kb to exon. A single loxp-site, containing an engineered -site for Southern Blot analysis, was inserted bp upstream of exon and a loxp/frt-flanked Neo cassette was inserted 0 bp downstream of exon. Thus the target region is bp long and includes exon. The figure is not exactly drawn to scale! BA (C7BL/ x 9/SvEv) hybrid embryonic stem cells were electroporated with 0µg of linearized targeting vector and selected with G8 antibiotic. Panel B. Screening and analysis of the retention of the third loxp-site. (Initial Screening was carried out by PCR, using reverse primer -ACGCCAGTGTCATGTTGTTG- downstream of the short arm and forward primer - GCATAAGCTTGGATCCGTTCTTCGGAC- within the Neo cassette generating a product of.7 kb in case of homologous recombination; data not shown). Four PCR-positive clones were expanded (indicated by an x) and retention of the third loxp-site upstream of exon was proven by PCR with primers -GTAACCATGATTAGTCTTTTAGCTTTATG- and - GTTCTGAGAATCCAAAGCACAAC- generating a bp long fragment from the WT-allele and an additional 9 bp long fragment from the floxed allele. Panel C. Southern Blot analysis. Further confirmation of the expanded homologous recombinant clones was performed by two different Southern Blot experiments. Left panel: -digested DNA was hybridized with a 7 bp long probe (probe primers: - TTTTGTGGCTAAGCACAGTCCC- and -CATACTGCAGCTCTGCTCAGATTC- ), which was targeted against the external region, detecting a 9. kb long WT-fragment and the.7 kb long floxed fragment. Right panel: -digested DNA was hybridized with a bp long probe (probe primers: -TGACTGCCCTCAACAGTTCATTTG- and -ATTCAGGATCTGCTACCACCACTG- ) targeted against the internal region, detecting a 9. kb long WT- and the. kb long floxed fragment. In both Southern Blot experiments, DNA from C7Bl/ (B), 9/SvEv (9), and BA (C7Bl/ x 9/SvEv; Hyb) mouse strains were used as wild type controls. Confirmation of Neo-deletion and standard Genotyping procedures. Targeted itl BA (C7BL/N x 9/SvEv) hybrid embryonic stem cells were microinjected into C7BL/ blastocysts. Resulting chimeras with a high percentage agouti coat color were mated to C7BL/ homozygous FLP mice to remove the Neo cassette. Primers Ndel ( -GTTGTGCTTTGGATTCTCAGAAC- ) and Ndel ( -CTTCTACCCTTCTATTTCAGGAAGC- ) were used to confirm Neo-deletion indicated by a 7 bp fragment. DNA still containing Neo is not amplified since the fragment would be too long. The same primer pair also generates a 97 bp long WT-fragment. Thus it can be routinely applied for standard genotyping. Genotyping primers are not depicted within the figure.
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