Anti γh2av

Protein extracts from Drosophila larval brains were obtained by dissecting 10 larval brains in 0.7% NaCl and homogenizing them in 20 µl of 2X Laemmli buffer. Protein samples were loaded into a 4-20% Mini-PROTEAN TGX precast gel to perform electrophoresis (SDS-PAGE) and blotted using the Trans-Blot® Turbo TM Transfer System on a nitrocellulose membrane (Hybond ECL, Amersham). Filters were blocked in 5% non-fat dry milk dissolved in 0.1% Tween-20/PBS for 30 min at RT and, then, incubated with anti-Giotto (1:5000; Rabbit; 59 ) and anti-γH2Av (1:1000; Mouse; Developmental Studies Hybridoma Bank, IA 52242) overnight at 4°C. The membranes were then incubated with HRPconjugated anti-Mouse and anti-Rabbit IgGs secondary antibody (1:5000; Amersham) for 1 hour at RT and then washed again 3 times with 0.1%Tween-20/PBS. The chemiluminescent signal was revealed through either SuperSignal TM West Femto or SuperSignal TM West Pico substrate (Thermo Scientific TM ) using the ChemiDoc scanning system (Bio-Rad). Band intensities were quantified by densitometric analysis using the Image Lab 4.0.1 software (Bio-Rad). WB was repeated independently at least three times.