Anti cytochrome c antibody clone 7h8.2c12

The cytochrome c assay was performed as described previously.^{57 (link)} Briefly, wild-type or bax^−/−/bak^−/− MEFs were permeabilized in digitonin-containing buffer (20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1250 mM sucrose and 0.05% (w/v) digitonin) and then incubated with peptides (10 μM, dissolved in DMSO) for 1 hour at 30°C before pelleting via centrifugation. The supernatant was retained (soluble fraction), and the pellet was lysed in Triton-X100-containing buffer (20 mM Tris pH 7.4, 135 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton-X100 and protease inhibitors) to generate the pellet fraction. Both soluble and pellet fractions were analyzed for cytochrome c by Western blotting using an anti-cytochrome c antibody (clone 7H8.2C12; BD Biosciences).

Cytochrome c release assays performed with digitonin-permeabilized MEFs were performed as described previously.^{41 (link)} For the cytochrome c release assay with the unpermeabilized U937 cells, 2×10⁶ cells were treated with peptides (10 μM) for 3 hours at 37°C before pelleting by centrifugation. Cell pellets were then resuspended in digitonin-containing lysis buffer (20 mM HEPES pH 7.2, 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 0.05% (w/v) digitonin (Calbiochem) supplemented with protease inhibitors (Roche)) for 45 minutes at 30°C to permeabilize the plasma membrane, then pelleted again. The supernatant was retained (soluble fraction) and the pellet resuspended in cell-lysis buffer (20 mM Tris pH 7.4, 135 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 1% (v/v) Triton X-100, 10% (v/v) glycerol supplemented with protease inhibitors (Roche)) and incubated on ice for 1 hour before centrifugation. This supernatant was then retained as the pellet fraction. Both soluble and pellet fractions were subsequently analyzed by Western blotting using an anti-cytochrome c antibody (clone 7H8.2C12; BD Biosciences).