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For western blot analyses, 50 ��g protein was subjected to SDS-PAGE under reducing conditions, transferred, and blotted with the anti-IRF4 antibody described above. For in?vitro studies, adipocytes were washed twice with DMEM and then incubated for 6?hr in serum-free DMEM containing 1% fatty acid-free BSA in the presence or absence of 10?��M isoproterenol. Conditioned medium was then analyzed for glycerol with the Free Glycerol Determination Kit (Sigma). Adipocytes were then rinsed with PBS and harvested in RIPA buffer. Enol The protein content of cell lysates was determined with the DC Protein Assay kit (BioRad). For ex?vivo studies, adipocytes were isolated from epididymal fat pads by collagenase digestion. Cells were resuspended in Krebs-Ringer HEPES (KRH) buffer at 2?�� 105 cells/ml. Cells were incubated in the presence or absence of 1?��M isoproterenol for 0�C2?hr at 37��C with gentle shaking. Glycerol and NEFA content of conditioned medium was determined as above. For in?vivo studies, mice were fasted for 4?hr and injected with isoproterenol (10?mg/kg body weight). Blood was collected from the tail vein before and 15?min after injection, and glycerol and NEFA content of find more serum was measured as above. Lipogenesis assays were performed as previously described (Wiese et?al., 1995). In brief, after serum starvation, cells were incubated in the presence or absence of 10 nM insulin for 15?min and incubated with 5?mM [14C]-glucose for 1?hr at 37��C. Cells were then washed three times with ice-cold PBS. Radiolabeled glucose incorporation into lipid was assessed by scraping cells into 1?ml PBS and shaking vigorously with 5?ml Betaflour Liquid Scintillation Fluid (National Diagnostics). After samples settled overnight, radioactivity partitioning into the organic phase was determined by scintillation counting. The protein content of cell lysates was determined with the DC Protein Assay kit. Unpaired two-tailed Student's t tests and one-way ANOVA learn more were used. p?