Nano lc 2

cindr^CA06686 fly heads were homogenized in lysis buffer (50 mM Tris pH7.4, 150 mM NaCl, 10% glycerol, 0.5% NP-40) containing complete protease inhibitor (Roche). Homogenized samples were subjected to centrifugation at 20,000 g for 20 min at 4°C. Supernatants were incubated in Chromotek-GFP-Trap Agarose Beads (Allele Biotechnology) in a rotator at 4°C. Cindr complex was eluted from the GFP-agarose beads and samples were subjected to nanoLC-MS/MS analysis with a nano-LC II (Thermo Scientific) coupled to Thermo Velos pro (Thermo Scientific) mass spectrometer as described in detail in Yoon et al. (2017) (link). For confirmation of Cindr interactions, Cindr:GFP was immunoprecipitated with anti-GFP from head homogenates (Elav-Gal4; UAS-cindr.GFP / + and Elav-Gal4 / Y; UAS-cindr.GFP/ +). Control flies (Ctrl) were Elav-Gal4/ + and Elav-Gal4/Y. 200 fly heads were grinded with a pestle in lysis buffer. After centrifugation at 15,000 g for 30 minutes to precipitate debris, brain lysate were collected and then incubated in Chromotek-GFP-Trap Agarose Beads (Allele Biotechnology) for 2–3 hours at 4°C. Beads were then centrifuged, washed with lysis buffer 6 times, and then 40ul of Lamelli buffer was added to samples for western blot. In Figure 1E, paired input (3%) and IP samples were run together for each of the two blots shown (Left & Right).