Rabbit anti dj 1

Whole snap frozen C57BL/6 or FEB mouse brains were placed in 4 ml cell lysis buffer (Cell Signaling) plus 50 mM iodoacetamide (to block free cysteine-SH). Brains were mechanically homogenized via mortar and pestle, and then subjected to lysis via sonication on ice for 2 min, alternating 10 s on 10 s off. Lysate was then spun down at 16,000g at 4°C in a tabletop centrifuge for 1 hour. The resulting supernatant was divided into aliquots, with addition of either 50 μl Protein G beads (Pierce) and 5 μg anti-goat IgG (Santa Cruz); or 50 μl Protein G beads and 5 μg anti-DJ-1 (Goat, Abcam). Samples were rotated at 4°C for 48 h, washed three times in lysis buffer plus 0.25 M NaCl. Laemmeli sample buffer (without reducing agents) was added and beads were boiled for 10 min. Samples were then run on a 15% non-reducing SDS-PAGE gel, transferred to a PVDF membrane, and probed for glutathionylation using an anti-glutathione antibody (anti-GSH, Millipore). Membranes were stripped by incubation in 0.1 M Tris-HCl pH 6.8, 2% SDS, and 0.11 M β-mercapto ethanol at 50°C for 30 min followed by three washes in TBST for 15 min each. Blots were then probed with rabbit anti-DJ-1 (Cell Signaling) in the same manner as described above.