Jazz mix drosophila medium

The genetic background W¹¹¹⁸ and wild-type Canton^S fly lines were maintained at 25°C on Jazz mix Drosophila medium (Fisher, Waltham, MA, USA) prepared according to the manufacturer’s instructions. Jazz mix medium consists of brown sugar, cornmeal, yeast, agar, benzoic acid, methylparaben, and propionic acid; however, specific concentrations of each component are not available. Prior to conjugation assays, flies were transferred to defined Bloomington Drosophila Stock Center (BDSC) cornmeal medium prepared without the addition of propionate (53 ). Bacterial strains used in this study were confirmed to grow on fresh BDSC cornmeal fly medium, and conjugation occurs on fly medium surfaces in the absence of flies. Fly incubations were conducted in a refrigerated incubator at 25°C under 12-h/12-h light/dark cycles and 70% humidity.

The ³⁵S-methionine incorporation was based on the protocol described in ref. 54 (link). Briefly, Jazz-Mix Drosophila medium (Fisher Scientific, cat. no. AS153) was supplemented with 100 μCi ³⁵S-methionine per ml of food (American Radiolabeled Chemicals, St Louis, USA; 1 mCi/37 MBq, ARS 0104A). Ten flies were transferred to each vial containing radioactive food. After 24 h of feeding, flies were transferred to nonradioactive food for 30 min in order to purge undigested ³⁵S-methionine radioactive food out of the intestines. Flies were then homogenized in 200 μM 1% SDS and heated for 5 min at 95 °C. Samples were centrifuged for 5 min at 13,000g at 4 °C and supernatant was retained. Proteins were precipitated by the addition of the same volume of ice-cold acetone and incubation on −20 °C for 1 h. Samples were then centrifuged at 13,000g for 15 min at 4 °C, supernatant was removed and the pellet was resuspended in 200 μl 4 M guanidine-HCl. An amount of 100 μl of each sample was used for scintillation counting, and the remaining 100 μl was used for Bradford assay to determine total protein concentrations. Counts per minute (CPM) measurements were normalized to total protein for each sample.