Western blot analysis of mouse brain tissue

Mouse brain tissues from two independent cohorts obtained from Case Western Reserve University and The University of Vermont (a kind gift from Dr. Y. Janssen-Heininger), were dissected into three sections, midbrain, cortex and cerebellum. Midbrain portions of the brains were snap frozen in liquid nitrogen. Prior to use, midbrains were homogenized by physical disruption using a pestle in a 1.5 ml Eppendorf tube with Cell Lysis Buffer (Cell Signaling) containing protease inhibitors (Roche). Tubes were incubated on ice for 15 min followed by sonication three times for 5 sec each. Samples were clarified by centrifugation at 4°C for 30 min at 16,000 x g. Aliquots of the supernatant (70 μg of protein content) were run on a 15% SDS-PAGE gel, transferred to PVDF membrane using a semi-dry transfer apparatus (Bio-Rad) at 20 V for 1 h, blocked with 5% milk in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h, and probed overnight with anti-actin (Sigma, diluted 1:5000), DJ-1 (Abcam,1:5000) and Grx1 (Genscript, 1:1000) antibodies. Blots were washed three times for 10 min each in TBST and probed with secondary antibodies (Bio-Rad, 1:5000) for 1 h. Blots were developed using enhanced chemiluminescence (Thermo Fisher). Rabbit polyclonal anti-Grx1 was generated by Genscript against recombinant human Grx1 produced in E. coli [ 20]. The western blots were scanned and band densities were quantified using ImageJ software (NIH).