Anti δn bcl xl

After protein purification, protein concentration was determined using BCA protein reagents (Thermo Scientific). Samples were separated on a 4–12% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA, USA) and probed with anti-Bcl-xL (1:1000 dilution, Cell Signaling), anti-ΔN-Bcl-xL (1:100 dilution, Aves Labs, Tigard, OR, USA), anti-active bax (1:100 dilution, Enzo Life Science, Farmingdale, NY, USA), anti-whole bax (1:1000 dilution, Cell Signaling), anti-cytochrome c (1:1000 dilution, Cell Signaling), anti-active caspase 3 (1:100 dilution, Abcam), anti-VDAC (1:1000 dilution, Cell Signaling), anti-COX IV (1:1000, Invitrogen) and anti-GAPDH (1:1000, Sigma-Aldrich). anti-ΔN-Bcl-xL is custom-produced (peptide sequence: CZ DSP AVN GAT GHS SSL D (1:100, Aves Labs). Membranes were treated with ECL reagents (Perkin Elmer) and images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Primary hippocampal neurons fixed in 10% buffered formalin were blocked in 10% goat serum for 1 h, then incubated with anti-HA (1:10 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GFP (1:100, 1:100, Abcam), anti-ΔN-Bcl-xL (1:10 dilution, Aves Labs), and anti-ATPB (1:100 dilution, Abcam) overnight at 4 °C. Cells were washed and incubated with Alexa-fluor 488 antibody or Alexa-568 antibody (1:200 dilution, Invitrogen, Molecular Probes, Carlsbad, CA, USA) for 1 h at room temperature and mounted on glass slides. Images were taken with a Zeiss Axiovert 200 microscope (Zeiss, Oberkochen, Germany) and processed using AxioVision 4.8.