Rabbit anti vacht

Synaptosomes were isolated from ventral and dorsal horn of fresh spinal cords using the Syn-PER Reagent (Thermo Scientifics). Synaptosomes were kept at 0-4 °C throughout the procedure and used for all assays. Antibodies used for characterization of synaptosomes were mouse anti-Lamin A/C, 1:1500 (Santa Cruz), rabbit anti-Synaptophysin, 1:5000 (Dako), rabbit anti-VAChT, 1:2000 (Sigma), goat anti-Na + /K + ATPase-α3 (sc-16052), 1:500 (Santa Cruz), mouse anti-GFAP, 1:1000 (Santa Cruz) and rabbit anti-SOD1, 1:2000 (Sigma), rabbit anti-VAChT, 1:2000 (Sigma), goat anti-Na + /K + ATPase-α3 (sc-16052), 1:500 (Santa Cruz). Synaptosomes were incubated with 1 % Triton X-100 for 20 min on ice and centrifuged at 14,000g for 15 min to obtain the supernatant. 60 µl of paramagnetic beads coated with G protein (Dynabeads; Invitrogen) were added to 200 μg of sample, and the mixture was precleared at 4 °C for 2 h. The mixture was centrifuged at 15,300g for 30 s and the supernatant was mixed with appropriate antibody and incubated at 4 °C overnight. 60 μl of anti-mouse or anti-rabbit IgG beads was added and incubated for 2 h, centrifuged at 15,300g for 30 s. The beads were recovered by centrifugation, washed thrice in PBS-1 % Triton X-100 buffer, resuspended in 2× Laemmli buffer, separated on 12 % SDS-PAGE gels and analyzed by IB.