Imager 600

Vectors expressing the editors (4 μg) and gRNA-puro (2 μg) were co-transfected into HEK293T cells in 6 cm dish (JETBIOFIL). Puromycin (InvivoGen) was added 24 h later to a final concentration of 2 μg/ml. Cells were harvested 72 h after transfection, and total protein extracted by RIPA lysis (EpiZyme). The protein samples were separated by SDS-PAGE, transferred to PVDF membrane (Merck Millipore). After blocking with 5% (w/v) non-fat milk dissolved in TBST (25 Mm Tris, PH 8.0, 150 Mm NaCl, and 0.1% Tween 20) for 1 h, the membranes were incubated overnight with anti-CRISPR-Cas9 antibody (Abcam # ab204448) or anti-GAPDH antibody (Absin #abs132004) at 4 °C. After extensive washing, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Proteins were visualized using Enhanced Chemiluminescence (ELC) reagent (Merck Millipore) and detected with an Amersham Imager 600.

Whole flies (ten per sample) were frozen at −20°C and lysed at 4°C in 300 µl of Laemmli Buffer supplemented with 1% Complete protease inhibitor (Roche) and NuPAGE™ Sample Reducing Agent (Thermo Fisher Scientific). Sample input was normalised on fly's weight. Proteins were separated by SDS-PAGE on 4–12% polyacrylamide gradient gels (ThermoFisher Scientific) and then electrotransferred onto nitrocellulose membranes (Bio-Rad). Standard immunochemistry protocols were used with primary rabbit anti-dFil (1/5000, provided first by Erika Geisbrecht and next by Boster Bio) and mouse anti-LaminC used as a loading control (LC28.26, 1/10,000, DSHB) and rabbit and mouse HRP secondary antibodies (Jackson ImmunoResearch Laboratories, ref 111-035-144, and ref 111-035-003 respectively, 1/50,000). Immunoreactivity was imaged and quantified with ImmobilonTM Western Chemiluminescent HRP Substrate (Merck Millipore) on an Amersham Imager 600 apparatus.