2.13. Co‐immunoprecipitation

Samples were homogenized in ice‐cold ‘immunoprecipitation buffer’ containing 20 mmol/L Tris‐HCl, pH 7.5, 150 mmol/L NaCl, and 1.5% Nonidet P‐40 supplemented with protease inhibitors. The extracts (500 µg protein per sample) were pre‐cleared with protein A/G beads and then mixed with non‐specific IgG (1 µg) or polyclonal mouse anti‐Bcl2 antibody (Cell Signaling Biotechnology), or anti‐CypD (Santa Cruz Biotechnology) overnight at 4°C, followed by the addition of 40 µL of protein A/G‐agarose (Santa Cruz Biotechnology) for 3 hours at 4°C. Immune complexes were washed four times in an ‘immunoprecipitation wash buffer’ (100 mmol/L Tris‐HCl, pH 7.5, 100 mmol/L NaCl, 0.1% Triton X‐100) and resuspended in 2× Laemmli buffer (Santa Cruz Biotechnology). The inputs were also mixed with 2× Laemmli buffer, and then the immunoprecipitation reactions and inputs were boiled for 10 minutes and pelleted in a centrifuge. The supernatants were subjected to Western blot analysis as described above.