Interactions between Mcl-1 and Bim or Bak were evaluated by co-IP analysis. CHAPS buffer (150 mmol/L NaCl, 10 mmol/L HEPES [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid] pH 7.4, protease inhibitors, and 1% CHAPS) was employed to avoid artifactual associations [43] (link). Cells were lysed in CHAPS buffer and 200 µg of protein per condition were immunoprecipitated with 1 µg anti-Mcl-1 (Santa Cruz Biotechnology or BD Biosciences), anti-Bak, or anti-Bim (Santa Cruz Biotechnology), followed by Dynabeads (Dynal, Oslo, Norway). IP samples were then subjected to Western blot analysis using anti-Bim (Millipore), anti-Mcl-1, or anti-Bak (Santa Cruz Biotechnology) as primary antibodies, respectively.
To monitor Bak and Bax conformational change, anti-Bax (6A7, Sigma) or anti-Bak (Ab-1, Millipore) antibodies, which only recognize Bax or Bak that have undergone conformational change, were used for IP, followed by Western blot analysis using anti-Bax and anti-Bak as primary antibodies.
To monitor Bak and Bax conformational change, anti-Bax (6A7, Sigma) or anti-Bak (Ab-1, Millipore) antibodies, which only recognize Bax or Bak that have undergone conformational change, were used for IP, followed by Western blot analysis using anti-Bax and anti-Bak as primary antibodies.