For RT-PCR, cDNA was synthesized from total RNA with the SuperScript III First-Strand synthesis system (Invitrogen) and subjected to Taqman RT-PCR on a ABI Prism 7900HT (Life Technologies).

Galectin-3 DNA primer sequ-ences were forward-CGGTCGTAGGTGAGCATCGTTGAC[FAM]G and reverse-CCCTTTGAGAGTGGCAAACCAT. Samples (n = 3 per group) were normalized to the relative amounts of reverse transcribed GAPDH, and expression levels calculated using 2.2 Sequence Inhibitors,research,lifescience,medical Detection Software (all from Applied Biosystems, Foster City, CA). Western blot, TNF-α, and protein carbonyl assays Spinal cord homogenates (n = 3 per group for Western blot, n = 4 per genotype for TNF-α and carbonyl assays) were prepared in Mammalian Protein Extraction Reagent (M-PER) buffer with protease inhibitors (Pierce Biotechnology, Rockford, IL) and protein concentrations determined with a BCA protein assay kit (Pierce Biotechnology). Proteins (15 μg) were resolved on 10% tris-glycine or bis-tris polyacrylamide gels and electrotransferred to Hybond ECL membranes, as previously described (Knoblach et al. 2004). Blots were Inhibitors,research,lifescience,medical blocked in blocking buffer (0.05% Tween 20, 5% milk powder in PBS) for 1 h, incubated with primary Inhibitors,research,lifescience,medical antibody for 1 h, washed 3× in 0.05% Tween in PBS, and then incubated with anti-HRP conjugated secondary antibody for 1 h. Blots were again washed 3× in 0.05%

Tween in PBS, and then developed with an ECL chemiluminescent detection kit (Amersham, Piscataway, NJ) and exposed to film. Primary antibodies included goat anti-mouse or anti-human galectin(s)-1, -3, and -9 (1:500; R&D systems, Minneapolis, MN) and antiactin (1:5000; Inhibitors,research,lifescience,medical Sigma–Aldrich, St. Louis, MO). Secondary antibodies included horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit (1:3000; Bio-Rad, Hercules, CA), or horseradish peroxidase-conjugated mTOR inhibitor donkey anti-goat (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA). For TNF-α, total protein (50 or 150 μg) was assessed in triplicate using a Multi-Analyte ELISA kit (SABiosciences, Rockville, Inhibitors,research,lifescience,medical MD). Protein carbonyls were detected with an OxiSelect Protein Carbonyl ELISA Kit (Cell Biolabs, San Diego, CA). Absorbance

was read at 450 nm. Immunohistochemistry Animals were perfused with PBS and 4% paraformaldehyde and spinal Fossariinae cords were removed, cryoprotected, frozen, and cut (20 μm). Sections (animal or human) were fixed in 4% paraformaldehyde (10 min), washed in PBS, and blocked in 5% donkey serum in 0.1% Triton X-100/PBS (1 h). Primary antibodies were applied (2 h, room temperature), sections were then washed in PBS, incubated with secondary antibody (1 h, room temperature), washed again, cover slipped, and viewed with a Bio-Rad MRC1024 confocal fluorescent microscope. Primary antibodies were as follows: goat anti-galectin-3 (1:500; R&D Systems), rabbit anti-GFAP (astrocyte marker; 1:1000; Sigma-Aldrich), and rabbit anti-IBA1 (microglial marker; 1:2000; Wako Chemical, Richmond, VA).