Akt pthr308

Sample preparation, western-blotting and quantification were performed as published by Trautenberg et al. (2019) (link). In brief, flies were raised on normal food and 5 days-old adults were transferred onto respective experimental foods for 7 days at 20°C. Thereafter, flies were snap-frozen in liquid nitrogen and frozen heads from females collected for processing. Heads were homogenized on ice in 0.01% Tx100-PBS and subsequently cooked at 95°C for 5 min. Polyclonal antibodies used to probe were Akt-pSer505 (Cell Signaling, 4054S), Akt-pThr308 (Invitrogen, 44-602G), Akt (Invitrogen, MAS14916).

mCherry::foxo flies were raised on normal food and 0 to 3-day-old adults were transferred for 14 days on respective diets at 20°C (male to female ratio 1:3; food renewal in regular intervals). Adults were snap-frozen with liquid nitrogen and fly heads removed. Fly heads were homogenized with a pestle in sample buffer (loading buffer, please see https://openwetware.org) and subsequently heat-inactivated for 10 min. Homogenates were centrifuged and supernatants used for TRIS-SDS-PAGE. Separated proteins were transferred by Tank-Blotting to nitrocellulose membrane. Membranes were blocked for 1 h with 5% BSA in 0.1% Triton X-100/PBS and then incubated with antibodies in blocking solution overnight. Polyclonal antibodies used to probe were Akt-pSer505 (Cell Signaling, 4054S), Akt-pThr308 (Invitrogen, 44-602G), Akt (Invitrogen, MAS14916), mCherry (Invitrogen, PAS-34974), and Tubulin (Cell Signaling, 2144S). After washing, membranes were incubated with HRP conjugated antibodies (Thermo Fischer, 31466) for 1 h and then bands detected with chemiluminescence.