Drosophila heads were homogenized in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10% glycerol, 50 mM NaF, 5 mM DTT, 4 M urea and Complete protease inhibitor cocktail (Roche Diagnostics)]. Proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose membranes (Whatman, Clifton, NJ, USA), blocked overnight in a 5% non-fat dried milk solution and probed with the following primary antibodies: rabbit anti-Drosophila TDP-43 (1:1500, made in-house), mouse anti-SYX 8C3s (1:2500, Developmental Studies Hybridoma Bank, DSHB, Iowa City, IA, USA), anti-CSP2c (1:9000, DSHB), mouse anti-tubulin CP06 (1:4000, Calbiochem, San Diego, CA, USA) and mouse anti-FLAG M2 (1:1000, Sigma-Aldrich) antibodies. The membranes were incubated with the secondary antibodies HRP-labeled anti-mouse-IgG (1:1000, Thermo Scientific, Rockford, IL, USA) or HRP-labeled anti-rabbit-IgG (1:1000, Thermo Scientific). Finally, protein detection was performed with Femto Super Signal substrate (Thermo Scientific) for anti- TDP-43 and anti-CSP2c immunoblotting and with ECL western blotting substrate (Thermo Scientific) for anti-syntaxin and anti-tubulin antibodies.
Protein expression was quantified using the NIH ImageJ software (Schneider et al., 2012 (link)) and normalized against tubulin. Histograms are representative of three independent experiments.
Protein expression was quantified using the NIH ImageJ software (Schneider et al., 2012 (link)) and normalized against tubulin. Histograms are representative of three independent experiments.