After the designated exposure to the choice or fixed diet for 2 or 3 weeks (food was changed every 2–3 days), experimental flies were quickly frozen, collected into groups of five, weighed, and then homogenized in 200 μl of cold phosphate-buffered saline containing 0.1% Triton X-100 (IBI Scientific) for 30 s at 30 Hz using a QIAGEN TissueLyser. For TAG quantification, the homogenate (20 μl) was added into 200 μl of Infinity Triglyceride Reagent (Thermo Electron Corp.) and incubated at 37°C for 10 min with constant agitation. TAG concentrations were determined by the absorbance at 520 nm and estimated by a known triglyceride standard. For protein measurement, 5 μl of fly homogenate was incubated with 200 μl of (1:50) 4% (w/v) cupric sulfate/bicinchoninic acid solution (Novagen) at room temperature for 30 min. Protein concentrations were estimated by the absorbance at 562 nm through the comparison with bovine serum albumin standards. Average weight, TAG, and protein values were based on at least six independent biological replicates (of five flies each) from multiple vials.

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