Cell signaling buffer

Neurons were scraped and lysed in 1X cell signaling buffer (Cell Signaling Technology, Danvers, MA, USA) and protein concentration was determined using BCA protein reagents (Thermo Fisher Scientific, Waltham, MA, USA). Samples (50–100 µg of protein/lane) were separated on a 4–12% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and probed with anti-Bcl-xL antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA) and anti-beta actin (Sigma-Aldrich, 1:1000). Scanned images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

After treatment of α-TCT and glutamate for 24 h, the primary hippocampal neurons were scraped and lysed in the 1X cell signaling buffer (Cell signaling Technology, Danvers, MA) and protein concentration was determined using BCA protein reagents (Thermo Scientific, Rockford, IL). Samples (50–100 µg of protein/lane) were separated on a 4–12% SDS–polyacrylamide gel (Bio-Rad, Hercules, CA) and probed with anti-∆N-Bcl-xL (1:100 dilution, Aves labs, Tigard, OR), anti-∆N-Bcl-xL (1:10, Pacific Immunology, Ramona, CA), anti-active caspase 3 (Abcam, 1:100), Bcl-xL (Cell signaling, 1:1000), and anti-beta actin (sigma, 1:1000). anti-∆N-Bcl-xL was custom-produced (peptide sequence: CZ DSP AVN GAT GHS SSL D. 1:100, Aves labs; 1:10, Pacific Immunology). Membranes were treated with ECL reagents (Perkin Elmer, Waltham, MA) and scanned with a C-DiGit blot scanner (Li-Cor, Lincoln, NE). Scanned images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).