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Effect of leucine supplementation http://www.selleckchem.com/products/DAPT-GSI-IX.html on Panc02 cell lines To confirm the proliferative effect of leucine supplementation seen in vivo, in vitro analyses were performed using the Panc02 cell line. To model the growth factor restrictive environment in CR mice relative to the overweight control mice as seen in Figure?1H, we grew the cells in media with either 1% FBS or 10% FBS. Supplementing media with 1% FBS has been used to mimic serum growth factor reduction found in calorie-restricted mice [33]. In the growth factor-rich environment of media with 10% FBS, cell viability was significantly increased by ~30% with 0.3 mM leucine supplementation (P Protein Tyrosine Kinase inhibitor on mTOR pathway intermediates In order to understand the differential response to leucine supplementation between the diet groups with respect to mTOR signaling, in vitro analyses were performed using Panc02 cell lines. Western blot analyses for the energy responsive intermediates p-AMPK, p-ACC, p-mTOR, p-p70S6K, and p-S6 revealed that the effects of leucine supplementation on cell signaling intermediates were impacted by growth factor availability. In the growth factor-rich environment of media with 10% FBS, supplementation with 0.3 mM leucine had no effect on phosphorylated AMPK, ACC, mTOR, p70S6K, or S6 ribosomal protein (Figure?5A,B). In the 1% FBS setting, leucine supplementation had no effect on phosphorylated AMPK or ACC, but did significantly increase phosphorylated mTOR (P NK cell and its downstream effector S6 ribosomal protein (P