Anti p eif2α antibody

A subcellular fractionation protocol provided by Thermo Scientific (Cat No. 78835; Waltham; MA) was used to isolate cytosolic and nuclear protein from cells. The protein concentrations of the cell lysates were measured using the Pierce BCA Protein Assay Reagent Kit (Cat No. 23225). Conditioned medium was collected from the supernatant by centrifugation for 5 min at 1500 rpm. Ten micrograms of proteins were separated by 15% SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was incubated with anti-HMGB1 antibody (Cat No. ab18256; Abcam), anti-HMGN1 antibody (Cat No. ab5212; Abcam), anti-p-eIF2α antibody (Cat No. ab32157; Abcam), anti-lamin A+C antibody (Cat No. ab108595; Abcam), or anti-β-actin antibody (Cat No. ab8227; Abcam) overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. The Promega Western Blot Detection System (Cat No. W1008; Madison; WI) was used to detect immunoreactive proteins.

Neurons receiving different treatments or tissues from the cerebral cortex were harvested with TNE buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol with protease inhibitors) or SDS lysis buffer (50 mM Tris, pH 8.0, 1% SDS). Lysates were centrifuged and processed for SDS-PAGE and transferred onto nitrocellulose memebranes. Western blot analysis was performed. Primary antibodies (anti-CDNF antibody 1:500, Sigma-Aldrich; anti-CHOP 1:100, Santa Cruz Biotechnology; anti-MANF antibody 1:500, Abcam, Cambridge, MA, USA; anti-eIF2α antibody 1:1000, Cell Signaling Technology; anti-p-eIF2α antibody 1:500, Abcam; anti-Tubulin 1:1000 and anti-actin 1:1000, Sigma-Aldrich) and horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000 dilution; Millipore) were utilized.