Briefly, mice were euthanized with an overdose of CO2 before cells were isolated from RptorEC−/−, RictorEC−/− or MtorEC−/− mice, and their wild-type littermates using positive immuno-selection with a rat anti-mouse CD31 (BD). EC purity was examined with the uptake of Dil-Ac-LDL (Biomedical Technologies) and FITC-conjugated anti-CD31 (Invitrogen) labeling. Primary purified aortic EC were cultured in EGM2 (Lonza)50 (link).
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