Phospho-mTOR (Ser2448) antibody (Cell Signaling Technology, #2971S), anti-phospho-p70 S6 kinase (Ser

Phospho-mTOR (Ser2448) antibody (Cell Signaling Technology, #2971S), anti-phospho-p70 S6 kinase (Ser371) antibody (Cell Signaling Technology, #9208), antimTOR antibody (Cell Signaling Technology, #2983), anti-p70 S6 kinase antibody (Santa Cruz, sc-230), anti-phospho-FoxO3a antibody (Upstate), anti-FoxO3a antibody (Upstate).Analysis of MitochondriaIsolation of mitochondria was performed as described previously [32]. In brief, mice were sacrificed by decapitation and their 10236-47-2 price livers were harvested immediately, washed in ice-cold isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA), and minced with a razor blade. Then the tissue was homogenized with a motorized Teflon/glass homogenizer, the homogenate was centrifuged for 5 minutes at 500 6 g at 4uC, and the supernatant was collected and re-centrifuged for 5 minutes at 500 6 g. The resulting supernatant was then centrifuged for 10 minutes at 8000 6 g at 4uC, and the pellet was suspended in isolation buffer. Unless otherwise indicated, all procedures were performed on ice. Protein concentrations were determined by the BCA protein assay (Pierce). Oxygen Lecirelin supplier consumption was measured with an Oxygen Meter (Model 781) and a Mitocell MT200 closed respiration chamber (Strathkelvin Instruments, North Lanarkshire, UK) at 37uC with continuous stirring in respiration buffer (125 mM KCl, 1 mM K2HPO4, 5 mM MgCl2, 25 mM HEPES, 0.2 mM EGTA, and 20 mM mannitol). Mitochondria, pyruvate, and malate (2.5 mM each), 500 nM rotenone, and 5 mM succinate were added sequentially to the buffer. Oxygen consumption by complex I was defined as the rotenone-sensitive component of oxygen consumption in the presence of pyruvate plus malate. Oxygen consumption by 1315463 complex II was defined as consumption after the addition of succinate minus consumption.RNA AnalysisTotal RNA was isolated from the livers of mice and from cultured cells with an RNeasy lipid tissue mini kit (Qiagen). For isolation of total RNA from C. elegans, an RNeasy MinElute Cleanup kit (Qiagen) was used. Real-time PCR was performed with a Light Cycler (Roche), the Taqman Universal Probe Library, and the Light Cycler Master (Roche) according to the manufacturer’s instructions. Pre-rRNA levels were evaluated by using specific primers for the external transcribed spacer, as described previously [30]. The copy number of mitochondrial DNA was assessed by quantification of a unique mitochondrial DNA fragment relative to a single copy region of the nuclear gene Tfrc (transferrin receptor) using real-time PCR [31].Isolation of HepatocytesHepatocytes were isolated as described previously [33,34]. In brief, 40 week-old mice were anesthetized and the abdominal cavity was opened. A 23G needle was introduced into the portal vein, and perfusion was started with Hepatocyte Liver Perfusion Medium (16) (Gibco) after proximal ligation of the inferior venaRole of Akt1 in LongevityRole of Akt1 in LongevityFigure 3. Ribosomal biogenesis and mitochondrial function in young and middle-aged female Akt1+/?mice. (A) Western blot analysis of phosphorylated mTOR, phosphorylated p70 S6 kinase, and phosphorylated FoxO3a expression in the livers of wild-type and Akt1+/?mice at 40 weeks old. (B) Pre-rRNA level in the livers of wild-type and Akt1+/?mice at 8 weeks and 40 weeks old were examined by real-time PCR (n = 10). (C) Mitochondrial DNA content of the livers prepared as Figure 2B. (D) Real-time PCR analysis of the expression of COX1 (encoding cytochrome c oxidase subunit I.Phospho-mTOR (Ser2448) antibody (Cell Signaling Technology, #2971S), anti-phospho-p70 S6 kinase (Ser371) antibody (Cell Signaling Technology, #9208), antimTOR antibody (Cell Signaling Technology, #2983), anti-p70 S6 kinase antibody (Santa Cruz, sc-230), anti-phospho-FoxO3a antibody (Upstate), anti-FoxO3a antibody (Upstate).Analysis of MitochondriaIsolation of mitochondria was performed as described previously [32]. In brief, mice were sacrificed by decapitation and their livers were harvested immediately, washed in ice-cold isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA), and minced with a razor blade. Then the tissue was homogenized with a motorized Teflon/glass homogenizer, the homogenate was centrifuged for 5 minutes at 500 6 g at 4uC, and the supernatant was collected and re-centrifuged for 5 minutes at 500 6 g. The resulting supernatant was then centrifuged for 10 minutes at 8000 6 g at 4uC, and the pellet was suspended in isolation buffer. Unless otherwise indicated, all procedures were performed on ice. Protein concentrations were determined by the BCA protein assay (Pierce). Oxygen consumption was measured with an Oxygen Meter (Model 781) and a Mitocell MT200 closed respiration chamber (Strathkelvin Instruments, North Lanarkshire, UK) at 37uC with continuous stirring in respiration buffer (125 mM KCl, 1 mM K2HPO4, 5 mM MgCl2, 25 mM HEPES, 0.2 mM EGTA, and 20 mM mannitol). Mitochondria, pyruvate, and malate (2.5 mM each), 500 nM rotenone, and 5 mM succinate were added sequentially to the buffer. Oxygen consumption by complex I was defined as the rotenone-sensitive component of oxygen consumption in the presence of pyruvate plus malate. Oxygen consumption by 1315463 complex II was defined as consumption after the addition of succinate minus consumption.RNA AnalysisTotal RNA was isolated from the livers of mice and from cultured cells with an RNeasy lipid tissue mini kit (Qiagen). For isolation of total RNA from C. elegans, an RNeasy MinElute Cleanup kit (Qiagen) was used. Real-time PCR was performed with a Light Cycler (Roche), the Taqman Universal Probe Library, and the Light Cycler Master (Roche) according to the manufacturer’s instructions. Pre-rRNA levels were evaluated by using specific primers for the external transcribed spacer, as described previously [30]. The copy number of mitochondrial DNA was assessed by quantification of a unique mitochondrial DNA fragment relative to a single copy region of the nuclear gene Tfrc (transferrin receptor) using real-time PCR [31].Isolation of HepatocytesHepatocytes were isolated as described previously [33,34]. In brief, 40 week-old mice were anesthetized and the abdominal cavity was opened. A 23G needle was introduced into the portal vein, and perfusion was started with Hepatocyte Liver Perfusion Medium (16) (Gibco) after proximal ligation of the inferior venaRole of Akt1 in LongevityRole of Akt1 in LongevityFigure 3. Ribosomal biogenesis and mitochondrial function in young and middle-aged female Akt1+/?mice. (A) Western blot analysis of phosphorylated mTOR, phosphorylated p70 S6 kinase, and phosphorylated FoxO3a expression in the livers of wild-type and Akt1+/?mice at 40 weeks old. (B) Pre-rRNA level in the livers of wild-type and Akt1+/?mice at 8 weeks and 40 weeks old were examined by real-time PCR (n = 10). (C) Mitochondrial DNA content of the livers prepared as Figure 2B. (D) Real-time PCR analysis of the expression of COX1 (encoding cytochrome c oxidase subunit I.