Rabbit anti mcl 1 antibody

Immunoprecipitation of active BAX and BAK was performed as previously described.^{52 (link)} Briefly, cells were lysed in CHAPS lysis buffer (10 nmol/l HEPES, pH 7.4; 150 nmol/l NaCl; 1% CHAPS). 500–1000 μg protein was immunoprecipitated and incubated overnight at 4 °C with 2 μg/ml mouse anti-BAK antibody (Ab-1; Merck Millipore, Billerica, MA, USA) or anti-BAX antibody (6A7, Sigma-Aldrich) and 10 μl pan-mouse IgG Dynabeads (Life technologies, Inc.), washed with CHAPS lysis buffer and analyzed by western blotting using rabbit anti-BAK antibody (BD Bioscience) or anti-BAX antibody (Merck, Darmstadt, Germany). Immunoprecipitation of MCL-1 was performed in 500 μl lysates containing up to 1000 μg proteins, which were incubated overnight at 4 °C with 2 μg/ml mouse anti-MCL-1 antibody (BD Biosciences) and 10 μl pan-mouse IgG Dynabeads or Protein G Dynabeads (Life Technologies, Inc.) and washed with CHAPS buffer. The precipitate was analyzed for interaction with NOXA by Western blotting using rabbit anti-MCL-1 antibody (ENZO, Lausen, Switzerland) and mouse anti-NOXA antibody (Alexis Biochemicals, Grünberg, Germany).

TUBE pull-down assay was performed as previously described.^{35 (link)} Cells were lysed in buffer (50 mM NaCl, 20 mM Tris pH 7,5, 1% NP40, 5 mM EDTA, 10% glycerol) supplemented with a protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany) for 20 min on ice. In all, 1000 μg of protein were incubated overnight at 4 °C with 50 μl GSH-agarose beads (Sigma-Aldrich) linked to GST-TUBE. Afterward beads were washed with buffer. The precipitate was analyzed for ubiquitin expression by Western blotting using mouse IgG1 anti-ubiquitin (P4D1) (Santa Cruz Biotechnology) antibody and for interaction with NOXA and MCL-1 by Western blotting using rabbit anti-MCL-1 antibody (ENZO), and mouse anti-NOXA antibody (ENZO). To verify the protein amount per lane, the membrane was stained with Ponceau S (AppliChem GmbH, Darmstadt, Germany).