Mouse anti cytochrome c monoclonal antibody

Cells were homogenized in buffer A (250 mM mannitol, 250 mM sucrose, 1 mM EDTA, 50 mM Tris-HCl, 1 mM DTT, 1 mM PMSF, 1 mM benzamidine, 0.28 U/l apotinin, 50 mg/l leupeptin, 7 mg/l pepstain A). Homogenates were centrifuged at 1000 × g for 10 min and the resultant supernatant was then centrifuged at 10 000 × g for 30 min, and the mitochondrial pellet was resuspended in buffer B (250 mM sucrose, 1 mM EGTA, 10 mM Tris-HCl). Supernatants were separated from the mitochondria by centrifugation at 100 000 ×  g for 1 h, and the supernatant was then used to analyze cytochrome c in the cytosol with immunoblotting. Total mitochondrial proteins and the postmitochondrial supernatants were electrophoretically separated on 12.0% SDS-polyacrylamide gels and transferred to 0.2-μm nitrocellulose membranes. The membranes were blocked with 5% (wt/vol) non-fat milk for 1 h, followed by incubation for another 1 h with mouse anti-cytochrome c monoclonal antibody (BD Biosciences Pharmingen, San Jose, CA, USA). The blots were then incubated with donkey anti-mouse IgG conjugated with horseradish peroxidase (Sigma). Enhanced chemoluminescent autoradiography (ECL Kit; Amersham, Arlington Heights, IL, USA) was used for the final detection. The mitochondrial preparation was also used for the determination of BAX protein levels with immunolotting.

Rabbit polyclonal antibodies against caspase-3, poly (ADP-ribose) polymerase (PARP), MCL-1, phospho-MCL-1 (T163), BIM, BAK, BAX, phospho-AKT (S437), DRP1, phospho-DRP1 (S616), OPA1, and TP53, and horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies against BCL-xL and mouse monoclonal antibodies against BCL-2, FIS1, BAX, NOXA, and β-actin were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit polyclonal antibodies against phospho-DRP1 (S637), MFF, and MUL1, and a mouse monoclonal antibody against Cox IV were obtained from Abcam (Cambridge, UK). Mouse anti-cytochrome c monoclonal antibody was obtained from BD Pharmingen (San Diego, CA, USA). Details regarding each antibody are presented in Table S3.

HeLa cells were plated onto 13 mm cover glasses (Assistant, 41001113) in a 24-well plate, transfected with 300 ng hBax-C3-EGFP plasmid and incubated with Q-VD-OPh for 16 h. Cells were then fixed with 4% paraformaldehyde in phosphate buffer saline (PBS), pH 7.2 for 30 min. The cover glasses containing cells were removed from the plate, blocked for 1 h in 10% goat serum (Sigma G6767) and 1% Saponin (Sigma 8047-15-2) and incubated overnight at 4°C with 1:250 mouse monoclonal anti-cytochrome c antibody (BD Pharmingen 556432) and 1:250 rabbit polyclonal anti-TOM20 antibody (Santa Cruz Biotechnology; sc-11415). The samples were then incubated with 1:200 goat anti-rabbit Alexa Fluor 405 and donkey anti-mouse Alexa Fluor 647 antibodies (Invitrogen; A31556 and A31571, respectively) for 1 hr at room temperature and mounted with ProLong Diamond Antifade Mountant (Invitrogen P36965) on an imaging slide. Imaging was performed on a Zeiss LSM 710 confocal microscope with a 63× Plan Apo oil-immersion objective with NA=1.4. Alexa Fluor 405, GFP-Bax, and Alexa Fluor 647 were excited at 405, 488, and 633 nm, respectively. Immunofluorescence images shown in Figure 1—figure supplement 1 have been adjusted for contrast individually.