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To the 10.000 rpm supernatant containing microsomes and cytosol fractions, 0.2 volume of 0.1 M calcium chloride solution was added, and further centrifuged at 10,000 rpm for 10 minutes to precipitate the microsomes

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Once activated, Akt phosphorylates tuberous sclerosis complex 2 (TSC2), suppressing the inhibitory effect of the TSC1-TSC2 complex in mTORC1