However, for the sake of simplicity, only the major repressor is included in our model

0 Hz for 30 min and stored at 220uC freezer overnight. The supernatants were collected after centrifugation at 10,000 rpm for 10 min. The remaining pellets were washed with 2 Aging and EETs 100:L of ice-cold methanol with 0.1 % 3838489 of acetic acid and 0.1% of BHT and centrifuged. The supernatants of each sample were Acacetin site combined and diluted with 2 mL of H2O and load onto SPE cartridges. Further sample preparation 21560248 was as described for plasma sample preparation. LC/MS/MS Analysis Liquid chromatography/tandem MS analysis of oxylipins was performed using a modified method based on the previous publication. An Agilent 1200 SL liquid chromatography series with an Agilent Eclipse Plus C18 2.1 x 150 mm, 1.8:m column was used for the oxylipins separation. The mobile phase A was water with 0.1% acetic acid while the mobile phase B was composed of acetonitrile/ methanol and 0.1% acetic acid. Gradient elution was performed at a flow rate of 250:L/min and the gradient used is described in appropriate HRP-conjugated secondary antibodies of anti-mouse or anti-rabbit were used at 1:1000 dilutions and developed using West Pico enhanced chemiluminescence. Proteins were normalized to GAPDH, which did not vary among groups. When separate gels were run for multiple samples, internal normalization controls were used in order to accurately compare the gels. Data analysis Results are presented as the mean +/2 SEM of at least three separate experiments. Data were analyzed by a one-way ANOVA or an ANOVA on Ranks, followed by a Student Neuman Keuls test or Dunn’s test, where appropriate. A p, 0.05 was considered significant. Results Plasma EETs levels did not differ based on either age or estrogen status. In contrast, the DHETs showed some marked differences. The 14,15-DHET levels in the Aged Ovx group were higher than the other treatment groups. The 5,6-DHETs in the aged OP group were lower than those measured in the adult OP group. The plasma 11,12- and 8,9-DHET levels did not differ among the groups. Three enzymes are reported as responsible for the majority of EETs synthesis – Cyp2J2, Cyp2C2, and Cyp2C6. Soluble epoxide hydrolase metabolizes EETs to DHETs and is the principal route of EETs metabolism. The major sources of EETs are the liver and kidney. As seen in Western blot analysis Analysis was performed as previously described. Prior to analysis, albumin was removed from the liver samples, as the abundant amount of this protein interfered with analysis of other proteins of similar size. Samples from a 3:1 mixture with Affi-gel blue were agitated for 30 minutes at 4u C and briefly centrifuged to remove the Affi-gel blue which is crosslinked to agarose beads. Generally, aging led to a decrease in Cyp protein levels, though an increase was seen in liver Cyp 2C6 level. sEH levels, by contrast, varied only in the kidney, based on estrogen status, with both OP groups showing increased amounts of sEH. The measured plasma levels of EETs, however, did not vary among groups. Details of elution gradient. doi:10.1371/journal.pone.0070719.t001 3 Aging and EETs EETs Synthesis EETs are formed by the metabolism of arachidonic acid by cytochrome P450 epoxygenases. In humans, the major Cyps reported to be responsible for EET synthesis are 2C8, 2C9 and 2J2, corresponding to Cyps 2C2, 2C6 and 2J2 in the rat. The principal sources of circulating EETs are reported to be the liver and kidneys. In the liver, aging led to decreased expression of Cyp2C2 and 2J2 and an increase in Cyp2C6. T

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