Femto supersignal substrate

Proteins were extracted from adult heads or from dissected larval brains. The material has been squeezed in lysis buffer 1 X (Lysis buffer composition 1.5 X: 225 mm NaCl, 15 mm Tris, 7.5 mm EDTA, 15% glycerol, 7.5 mm EGTA, 75 mm NaF, 6 M urea, 7.5 mm DTT and protease inhibitor) and then clarified by a short centrifugation at 0.5 × g. The proteins were separated by SDS-PAGE and blotted on 0.2 µm nitrocellulose membrane (Whatman Protran). Membranes were blocked overnight in 5% nonfat dried milk in TBS-0.01% Tween 20 and probed with primary antibodies: anti-TBPH (in house 1:2000) and anti-Tubulin (Calbiochem 1:4000). Goat anti-rabbit and goat anti-mouse IgG HRP conjugated were used as secondary antibodies (Thermo Scientific 1:1000). Detection was done with Femto SuperSignal substrate (Thermo Scientific). Band intensity was quantified using ImageJ and normalized on loading control. In every lane the mean percentage obtained out of at least three experiments has been shown and normalized to control genotype.

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.