Co-immunoprecipitation

For co-immunoprecipitation experiments, Drosophila Karst YFP knock-in embryos (DGRC 115285), Wiso embryos, and embryos expressing Patronin–GFP or Shot–GFP were collected over 24 h at 22°C before being lysed in buffer containing 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 and 0.5 mM EDTA (Chromotek), plus PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche), protease inhibitor cocktail (Roche), 0.1 M NaF and 1 mM PMSF. Samples were left on ice to solubilise for 30 min, before being centrifuged at high speed (14,000 rpm in a desktop centrifuge for 30 min at 4°C). The supernatant was collected, pre-cleared and incubated with GFP Trap-M beads (Chromotek).

Western blots were probed with mouse anti-GFP, guinea pig anti-Shot, rabbit anti-Patronin, mouse anti-α-Spectrin and rabbit anti-βH-Spectrin antibodies (details in Table S3; see Fig. S4 for complete western blots and for siRNA knockdown experiments in human cells), before being detected with chemiluminescence (GE Healthcare).