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Ethanol (2?g/kg) was administered and adrenals were collected after 60?min. (a) Adrenal TSPO (18?kDa) and (b) MLN64 (53?kDa) levels were measured by western blot analysis. (c) Rats were pre-treated with the TSPO (18?kDa) antagonist PK11195 (1?mg/kg, i.p.) 30?min prior to ethanol administration and http://www.selleckchem.com/ steroid levels were measured 30?min post-ethanol. Protein levels were normalized to b-actin and presented as % control *p?<?0.001 compared to control, (anova followed by Newman�CKeuls test), n?=?6�C8 for each group in duplicate. Figure S2. Cycloheximide (CHX) administration following ethanol arrests ethanol-induced increases in StAR as well as plasma and brain steroid levels. Cycloheximide (20?mg, i.p.) was administered for the final 20?min of a 60-min ethanol exposure. (a) Adrenal StAR expression was normalized to b-actin and presented as % control. (b) Plasma progesterone levels, and (c) 3a,5a-THP levels in cortex and hippocampus. *p?<?0.001 compared to control, @p?<?0.05 compared to control and #p?<?0.01 compared to ethanol (anova followed by Newman�CKeuls tests), n?=?6 in duplicate. Figure S3. Immunoprecipitation of phosphorylated StAR protein. Adrenal mitochondrial fractions were prepared and (a) the specificity of phospho-PKA substrate antibody was tested by incubation of adrenal fraction (30?mg) with l-phosphatase (50?000 units) to dephosphorylate all proteins (Lane 1), or phosphatase inhibitor (1?:?100, Lane 2). (b) Adrenal mitochondrial fractions were solubilized in RIPA buffer and run on SDS�CPAGE to show that StAR protein is not bound to other proteins. Lane 1 shows heat denatured protein following <a href="http://www.selleckchem.com/products/MLN-2238.html">MLN2238 nmr RIPA solubilization, lane 2 shows RIPA solubilization without heat and lane 3 shows the adrenal fraction denatured in SDS with no RIPA solubilization. (c) Phospho-PKA substrate antibody immunoprecipitated phosphorylated StAR. Lane 1 shows the negative control demonstrating that IgG antibody from the same species does not immunoprecipitate StAR, lane 2 shows phospho-PKA substrate antibody immunoprecipitate of phosphorylated StAR and lane 3 shows a positive control for adrenal StAR protein. Figure S4. Inhibition of P450scc by aminoglutethimide (AG) attenuates ethanol-induced increases in plasma steroids. Rats were administered aminoglutethimide (20?mg, i.p.) or propylene glycol (PG) vehicle 60?min prior to ethanol or Azastene saline vehicle and tissue was collected 60?min post-ethanol administration. (a) Adrenal StAR protein levels were measured by western blot analysis. StAR levels are normalized to b-actin and presented as % control. (b) Plasma progesterone levels. *p?<?0.001 compared to control and #p?<?0.001 compared to ethanol, (anova followed by Newman�CKeuls test), n?=?6 in duplicate. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset.</div>
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