Blocking buffer

After protein samples were denatured at 70°C for 10 min under reducing conditions, proteins were resolved with 4–12% gradient 1D-SDS-PAGE (Invitrogen, Carlsbad, CA), and transferred to 0.45 µm nitrocellulose (or PVDF) membranes (Bio-Rad, Hercules, CA) using a Bio-Rad semi-dry transblotter. The membranes were blocked in LiCor blocking buffer (LiCor, Lincoln, NE) and then incubated overnight at 4°C in LiCor blocking buffer with 0.1% Tween 20 and the primary antibodies listed in Table 2. All antibodies were individually optimized to determine ideal conditions within the linear range of detection for each assay, and that the primary antibody was present in excess. After antibody incubations, the membranes were rinsed in phosphate-buffered saline with 0.1% Tween (PBST) and probed with IR-dye labeled secondary antibodies (Li-Cor; 1:10,000) for 1 h at RT. The membranes were rinsed again with PBST and then with deionized water. Immunoblots were scanned using a LiCor Odyssey near-infrared scanner using the Odyssey V3.0.16 software package.

Western blot analysis was performed according to our previously published techniques (Bauer et al., 2008 (link)). Briefly, homogenized human and mouse brain (fresh frozen) from frontal cortex was prepared for western blot analyses with double distilled filter purified water (dH₂0) and sample buffer (4.5% sodium dodecyl sulfate (SDS), 15% βmercaptoethanol, 0.018% bromophenol blue, and 36% glycerol in 170mM Tris-HCl, pH 6.8) and heated at 70C for 10 minutes. Samples were run on 4–12% gradient gels and transferred to polyvinylidene difluoride (PVDF) membranes using a semi-dry transblotter (Bio-Rad, Hercules, CA, USA). The membranes were blocked with LiCor blocking buffer (LiCor, Lincoln, NE, USA) for 1 hour at room temperature, and then probed with the primary antibodies. After three 8 minute washes in phosphate buffered saline (PBS), the membranes were then incubated with the appropriate second antibody with infared-Dye 670 or 800cw labeled in LiCor blocking buffer or 5% bovine serum albumin in PBS for 1 hour at room temperature. Washes were repeated after the secondary antibody incubation. Membranes were scanned using a LiCor Odyssey scanner, and the intensity value for each protein band was measured using the Odyssey 2.1 software.

Due to limited amounts of material from our fractionation experiment, we were only able to run 13 pairs of subjects for the EAAT2 exon 9 skipping and 12 pairs for the Na⁺/K⁺ ATPase β studies. Samples for western blot analyses were prepared with milli-Q water and sample buffer (6 × solution: 4.5% sodium dodecyl sulfate (SDS), 15% β-mercaptoethanol, 0.018% bromophenol blue, and 36% glycerol in 170 mM Tris–HCl, pH 6.8) and heated at 70 °C for 10 min. For protein analysis of subcellular fractions, the same amount of protein (5–10 μg) was loaded for each subject pair (Hammond et al., 2012 (link)). Samples were then run on 4–12% gradient gels and transferred to PVDF membranes by BioRad semi-dry transblotters (Bio-Rad, Hercules, CA, USA). The membranes were blocked with LiCor blocking buffer (LiCor, Lincoln, NE, USA) for all antibodies except EAAT2B which was blocked with 5% BSA for 1 h at room temperature, then probed with the primary antibodies. After three 8 min washes in 1 × PBS, the membranes were then incubated with the appropriate second antibody with IR-Dye 670 or 800cw labeled in LiCor blocking buffer or 5% BSA in PBS for 1 h at room temperature. Washes were repeated after secondary antibody incubation. Membranes were scanned using a Li-Cor Odyssey scanner, and the intensity value for each protein band was measured using the Odyssey 2.1 software.

For Western blot analyses, the tail nerve, DRG and spinal cord tissue was homogenized in 50 (nerve) or 100 μl (DRG and spinal cord) lysis buffer (10 mM Tris base, 2 mM EDTA, 150 mM sodium chloride, 1% Triton-X 100, and 1 μl/ml Sigma Protease inhibitor cocktail^™), and protein concentrations were measured using Bio-Rad _DC assay (Bio-Rad Inc.). The homogenates were diluted with Laemmli buffer (1:1.5), separated on a 10% SDS–PAGE gel, and transferred to nitrocellulose membranes. The membranes were blocked in LI-COR blocking buffer (LI-COR Biosciences, Lincoln NE) and probed for nitrotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA) or Beta-actin (Cell Signaling Technologies, Danvers, MA). The detection was performed with IRDye800-conjugated anti-mouse, or anti-rabbit IgG, respectively in LI-COR blocking buffer. At the end of incubation period, the membranes were washed 3× with TBST. The membranes were visualized and analyzed on Odyssey IR imaging system (LI-COR Biosciences, Lincoln, NE). Band signal intensities are presented as area intensity relative to control samples after normalization to beta actin as loading control.

Protein analysis was performed as described previously [29 (link)] on whole hippocampal samples of all treatment groups. Briefly, 20μg of protein were resolved in AnyKD™ precast polyacrylamide gel (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to nitrocellulose membranes. Membranes were incubated with LI-COR blocking buffer (LI-COR, Lincoln, NE, USA) for 1h at room temperature before reacting overnight at 4°C with primary antibodies: Neuregulin-3 (NRG3) (1:500, PA5–18552, Invitrogen, Carlsbad, CA), ErbB4 (1:500, NBPI-33120, Novus, Centennial, CO.), and Beta-tubulin (1:2000, 2128L, Cell Signaling Technology, Danvers, MA.). After washing in phosphate buffered saline-Tween-20, the blots were incubated in fluorescent secondary antibodies (1:20000, LI-COR) in LI-COR blocking buffer for 1 h at room temperature. Membranes were then washed, and immunolabeling detection and densitometry measurements were performed using the LICOR Odyssey System (LI-COR). Ratios of the proteins of interest (NRG3 and ErbB4) to the housekeeping protein (β-actin) densities were calculated for each sample and normalized to 4-week old saline-treated controls.