The cell lysate was incubated on a shaker for 30 minutes at 4 C and centrifuged at 15000 g for 15 min. 1 ml supernatant was incubated with 30 ul sus pended ANTI FLAG M2 affinity gel overnight at 4 C and then spun at 15,000 g for 15 min. The M2 resin was washed 4 times with buffer A, bound proteins were eluted using buffer B. Eluted proteins were separated by elec trophoresis using 4 20% gradient PAGE gels. In order to disrupt the non covalent interactions, cells were lysed with lysis buffer containing 1% sodium dodecyl sulfate and then heated to 100 C. 10 mM sulfhydryl alkylating agent N ethylmaleimide was added into the cell lysis buffer to prevent ubiquitin chain from breaking. The concentrations of SDS in the cell lysis were diluted to 0. 1% before immu noprecipitated with the ANTI FLAG M2 beads.
Background Fish are important mTOR activation components of the human diet, being highly nutritious and valued as the main source of n 3 long chain polyunsaturated fatty acids . These essential fatty acids, mainly eicosapentaenoic acid and docosahexaenoic acid, have well known health promoting properties, including protec tion against a range of cardiovascular and inflammatory diseases, and neurological disorders. With population growth and increasing awareness of the importance of fish consumption as part of a healthy diet, worldwide de mand for seafood continues to grow. However, as trad itional fisheries are largely in decline, aquaculture must meet this demand. Aquaculture is the fastest growing food production sector with an average annual growth rate of 6. 6%, accounting for 46% of total fish supply.
In selleck chemicals the European and American continents, aquaculture production is largely dominated by salmonid species, mainly Atlantic salmon, and feeds for such carnivorous species have traditionally relied on fishmeal and fish oil from wild stocks. Recent estimates indi cated that 88. 5% of global production of FO was used by the aquaculture sector, with salmonid culture taking the largest share. With ever increasing demands for aquafeeds and reduction in fisheries landings, the availability of FO and FM seriously limits the growth of aquaculture production and there is an urgent need to find more sustainable alternatives. Vegetable oils can replace FO in salmon feeds without compromising fish growth or condition al though, at high levels of replacement, tissue levels of n 3 LC PUFA are significantly reduced. The effects of FO replacement by VO are becoming well characterized in the hepatic transcriptome of salmonids, and other species. However, studies on intestinal tran scriptome are few and restricted to effects of replace ment of FM by plant proteins, particularly soybean meal, given its potential to cause enteritis.