Mouse anti caspase 3

Protein measurements were carried out using the Pierce BCA Protein Assay Kit (Thermo Scientific) in accordance with the manufacturer’s instructions. Samples were mixed with Laemmli’s loading buffer, boiled for 5 min, and subjected to SDS-PAGE (12%) followed by blotting onto nitrocellulose membranes for 30 min at 25 V using the Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked for 1 hr with 5% non-fat milk in TBS at room temperature and subsequently probed overnight at 4°C with the primary antibody (1:1000). The following primary antibodies were used: rabbit anti-vinculin, rabbit anti-hexokinase-II, rabbit anti-Na⁺/K⁺-ATPase, rabbit anti-cleaved caspase-3 (all from Cell Signaling), mouse anti-caspase-2, mouse anti-caspase-3, mouse anti-PARP1, mouse anti-cytochrome c (all from BD Transduction Lab), mouse anti-lamin B, rabbit anti-Endonuclease G, mouse anti-AIF (all from Santa Cruz Biotech), mouse anti-caspase-8 (Enzo Life Science), rabbit anti-GAPDH (Trevigen), and rabbit anti-ERp29 (kindly provided by Dr. S. Mkrtchian, Karolinska Institutet). After four times washes in TBST (0.05% Tween-20 in TBS), membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies purchased from Cell Signaling (1:4000) for 1 hr at room temperature. Blots were developed using ECL (Amersham Biosciences) and documented using Chemi-Doc (Bio-Rad).