Phospho akt ser505

Antibodies for Akt (#9272), phospho-Akt (Ser505) (#4054), non-phospho-4E-BP1 (Thr46) (#4923), phospho-4E-BP1 (Thr37/46) (#2855) and β-actin (#4967) were purchased from Cell Signaling Technology (Danvers, MA, USA). Ten flies were homogenized in 100 μl RIPA buffer with protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and PhosSTOP phosphatase inhibitor cocktail (Roche, Nutley, NJ, USA). Supernatant was incubated with LDS loading buffer (Invitrogen, Grand Island, NY, USA) at 70°C for 10 min. Thirty micrograms of denatured protein was separated on 10% NuPAGE Novex 4–12% Bis-Tris precast polyacrylamide gels (Invitrogen, Grand Island, NY, USA) and transferred to PVDF membranes. Following incubation with primary and secondary antibodies, blots were visualized with Pierce ECL western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA). Three independent biological replicates were generated for all analyses, and samples for AKT and 4E-BP produced at different times. Band intensity was quantified with Image Lab software (Bio-Rad, Hercules, CA, USA).

To assess insulin sensitivity, fat bodies (abdominal carcasses with the ovaries and digestive tract removed, n = 5 per sample) were dissected from live females anesthetized on ice and pooled into 1 mL Schneider’s medium (GIBCO 21720024). Samples were incubated for 15 min at 25°C, then treated ± 5 μM insulin (Sigma I9278) for a further 15 min at 25°C. The supernatant was removed and the fat bodies were homogenized into Laemmli buffer (BioRad #1610737) with 5% v/v β-mercaptoethanol. Samples were analyzed by western blotting, probing for phospho-AKT-Ser505 (1:1,000; Cell Signaling #4054) with total AKT (1:1,000; Cell Signaling #9272) and actin (1:1,000; Abcam #ab8224) as controls. Bands were analyzed by densitometry using FIJI software (ImageJ; Schindelin et al., 2012 (link)). To measure glucose uptake, dissected adult fat bodies (n = 5 per sample) were incubated in 100 μL Schneider’s medium with 200 μM 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; Invitrogen N13195) ± 5 μM insulin for 15 min at 25°C, protected from light. Samples were washed twice, then homogenized in 120 μL PBS using a pellet pestle and motor. 100 μL of supernatant was loaded in a 96-well plate, and the fluorescence (excitation/emission 485/520 nm) measured in a plate reader (FLUOstar Omega, BMG Labtech) against a 2-NBDG standard curve.