Rabbit anti mnsod

Rabbit anti-human GCDH antibody was kindly provided by Dr. S. I. Goodman (University of Colorado Health Sciences Center, Denver). The polyclonal mouse anti-human DLST and rabbit anti-human ETFA antibodies were purchased from Sigma (Munich, Germany), rabbit anti-human ETFB from Abcam (Cambridge, UK), and rabbit anti-LC3 from Abgent (San Diego, USA). The monoclonal mouse anti-GFP antibody was obtained from Roche (Mannheim, Germany) and rabbit anti-MnSOD from Millipore (Billerica, USA). Peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG was from Dianova (Hamburg, Germany). HRP-conjugated anti-V5 antibody, monkey anti-mouse IgG coupled to Alexa Fluor 488 and goat anti-rabbit IgG coupled to Alexa Fluor 546 were from Invitrogen (Karlsruhe, Germany).

To quantify nitrosylated MnSOD, nitrotyrosine-containing proteins were immunoprecipitated from 500 µg of cortex protein in immunoprecipitation buffer (20 mM Tris-base, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 2 mM EGTA, 1% protease inhibitor cocktail) with 10 µg of protein G sepharose beads, 2 µg anti-nitrotyrosine monoclonal antibodies (Millipore, Cat No. 05-233) washed in PBS (pH 7.4). Immunoprecipitates were resuspended in 40 µL of sample buffer (2X, 0.5 M Tris–HCl (pH 6.8), 2.5% glycerol, 0.5% SDS, 200 mM 2-mercaptoethanol, 0.001% bromophenol blue) and resolved in 12% SDS-PAGE separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked 3% bovine serum albumin (BSA), 0.01% Tween-20 (TBS/T) in Tris buffered saline, followed by incubation with rabbit-anti-MnSOD (1:1000; Millipore Cat No. 06–984). Membranes followed by horseradish peroxidase coupled secondary antibody (1:5000; Cell Signaling #7074) and visualized by enhanced chemiluminescence (ImageQuant LAS 4000, GE Healthcare). Total lysate was run in parallel and β-actin (1:5000; Sigma, Cat No. A5441) in these samples was for normalization. Protein bands were quantified by densitometry using ImageJ software.