Western Blot

Samples (cells or spinal cord tissue segments at L1-L6) were collected and washed with ice-cold PBS before being lysed in radio immunoprecipitation assay (RIPA) lysis buffer and then sample lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 10 % low-fat dry powdered milk or with 5% BSA and 5% low-fat dry powdered milk in TBST (Tris–HCl, NaCl, Tween 20) for 2 h at room temperature, and then probed with primary antibodies at 4°C overnight. Finally, the horseradish peroxidase (HRP)-coupled secondary antibodies were utilized for detecting corresponding primary antibody. The primary antibodies utilized included β-actin (1:5000), Caspase-12 (1:1000), HSP70 (1:1000), GRP78 (1:500), ATF6 (1:1000), p-eIF2α (Ser51) (1:1000), eIF2α (1:1000), IRE1α (1:1000), XBP1s (1:1000), ERK (1:1000), p-ERK (Thr202/Tyr204) (1:1000), PKA (1:1000), p-PKA(Thr197) (1:1000), NMDA Receptor subunit 1 (NR1) (1:1000), and Phospho-NMDA Receptor subunit 1 (p-NR1) (Ser897) (1:1000). The bands were then developed by enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, United States). Data were analyzed with the Molecular Imager and the associated software Image J (NIH, United States). The original unprocessed images of all western blots were provided in Supplementary Data Sheets S1–S10.