After exposure to the choice or fixed diet for 20 days, experimental flies were flash frozen in liquid nitrogen. Heads and bodies were separated using a metal sieve on dry ice, and 10 heads were pooled for each biological replicate. Heads were first pulverized to a fine powder using a plastic pestle on dry ice. Protein extraction was carried out on ice using RIPA buffer (Sigma–Aldrich) supplemented with protease inhibitor cocktail (Sigma), phosphatase inhibitor cocktail (Sigma), sodium orthovanadate (NEB, 1 mM), and sodium fluoride (NEB, 1 mM). Ice cold buffer (105µl) was added to heads followed by immediate homogenization with a motorized pestle for 10 s on ice. Lysates were incubated on ice for 10 min followed by 15 s of sonication and centrifugation at 16,000 × g at 4°C for 10 min. Supernatants were recovered, and protein concentration was determined using the Pierce BCA Protein Assay Kit (Fisher Scientific). Thirty micrograms of protein lysate was added to 2× protein sample buffer (1 mM Tris–HCL pH 6.8, 10% sodium dodecyl sulphate [SDS], 1% bromophenol blue, and 1M dithiothreitol) and denatured at 95°C for 10 min. Protein was separated by SDS–polyacrylamide gel electrophoresis on a 4–12% gel (Bio-Rad) at 200 V for 30 min, followed by electrophoretic transfer to PVDF membrane at 70 V for 1 hr. Blots were incubated in 5% milk in 1% TBS-T at room temperature for 1 hr, followed by overnight incubation with primary antibodies overnight at 4°C (anti-dS6K gift from Thomas Neufeld; dilution at 1:3500, anti-pS6KThr398 Cell Signaling Technologies, #9209; dilution at 1:1000, anti-GAPDH; dilution 1:6000). Membranes were washed with 1% TBS-T and incubated with HRP-conjugated secondary antibodies (Abcam) at room temperature for 4 hr. Membranes were washed again with 1% TBS-T and then incubated briefly in ECL substrate (SuperSignal West Femto, ThermoFisher) before imaging.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.