Biorupter plus

Mixed-stage animals grown under Hygromycin B (250 µg/ml) selection were washed off with M9 buffer (0.22 M KH₂PO₄, 0.42 M Na₂HPO₄, 0.85 M NaCl, 0.001 M MgSO₄), and washed two more times with M9 buffer. Samples were then pelleted and resuspended in 100–400 µl of Lysis Buffer (25 mM Tris HCl pH 7.5, 150 mM NaCl,1 mM EDTA, 0.5% Igepal 630, and 1 tablet/50 ml complete protease inhibitor cocktail (Sigma-Aldrich)). After flash freezing in liquid nitrogen and thawing, samples were sonicated with a Diagenode BioRupter Plus, 5 min high setting: 30 s on/30 s off in a 4 °C water bath. The lysates were then spun at max speed in a tabletop centrifuge at 4 °C for 15 min to clear cellular debris. ELISA was performed as above, but the SuperSignal ELISA pico chemiluminescent substrate was used undiluted (ThermoScientific).

Biotin (Thermo Scientific) was resuspended in PBS at a concentration of 0.25 mg/ml. 5 mls was added to cells in a 10 cm dish and incubated at 4 °C on a shaking incubator for 40 min. For each sample, after 40 min, 500 µl of quenching solution was added to the biotin supernatant and the entire supernatant was then removed from the cells and put into a 50 ml centrifuge tube. Cells were incubated in 6 mls of quenching solution on ice for 5 min before being scraped from the plate and collected in the same 50 ml centrifuge tube along with the biotin solution. Cells were pelleted and then washed with TBS. Afterward the cells were resuspended in 500 μl of ELB. Lysate was then sonicated using the Diagenode Biorupter Plus. Settings of the Biorupter Plus were as follows: low power, 3 × 5 s bursts with a 5 s delay in-between. After sonication, lysates were incubated on ice for 30 min and vortexed every 5 min for 5 s. Afterward, lysates were centrifuged at 10,000g for 2 min at 4 °C (Tomy MX-305 high speed centrifuge). The lysates (500 µg of protein) were incubated with NeutrAvidin agarose resin (Thermo Fisher Scientific) and rotated at 4 °C overnight. The NeutrAvidin agarose resin was then washed three times in ELB buffer and separated out on SDS-PAGE gels. Immunoblotting for proteins of interest was then performed.