Antibodies used were as follows: anti-synaptophysin, anti-ChAT, Anti-PSD95, anti-NeuN (Cell Signaling Technology, Beverly, MA); anti-GPX4 (Santa Cruz Biotechnology, Dallas, TX); anti-4-HNE antibody (R&D Systems, Minneapolis, MN); and anti-β-actin antibody (Abcam, Cambridge, MA).
Tissues were homogenized in RIPA buffer (20 mM Tris, pH 7.4, 0.25 M NaCl, 1 mM EDTA, 0.5% NP-40, and 50 mM sodium fluoride) supplemented with protease inhibitors (539,134, EMD Biosciences Inc., San Diego, CA). Levels of specific proteins in tissues were determined by western blots as previously described44 (link). In brief, 30 µg total protein per sample was separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% BSA then incubated with primary antibody overnight at 4 °C. After incubation with fluorophore-conjugated secondary antibodies (ThermoFisher, MA) for 1 h, bands were detected using an Odyssey scanner (LI-COR, Lincoln, NE). Alternatively, after incubation with primary antibodies, the membranes were incubated with a HRP-conjugated secondary antibody. The bands were visualized using the ECL Kit (RPN2132, GE Healthcare, Piscataway, NJ). The bands were quantified using NIH ImageJ software (ImageJ 1.52a;http://imagej.nih.gov/ij ) and normalized to the loading control (β-Actin). The experiments were repeated at least once.
Tissues were homogenized in RIPA buffer (20 mM Tris, pH 7.4, 0.25 M NaCl, 1 mM EDTA, 0.5% NP-40, and 50 mM sodium fluoride) supplemented with protease inhibitors (539,134, EMD Biosciences Inc., San Diego, CA). Levels of specific proteins in tissues were determined by western blots as previously described44 (link). In brief, 30 µg total protein per sample was separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% BSA then incubated with primary antibody overnight at 4 °C. After incubation with fluorophore-conjugated secondary antibodies (ThermoFisher, MA) for 1 h, bands were detected using an Odyssey scanner (LI-COR, Lincoln, NE). Alternatively, after incubation with primary antibodies, the membranes were incubated with a HRP-conjugated secondary antibody. The bands were visualized using the ECL Kit (RPN2132, GE Healthcare, Piscataway, NJ). The bands were quantified using NIH ImageJ software (ImageJ 1.52a;
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