Ab186321

Whole cell protein samples were sonicated and boiled in RIPA buffer and total protein was quantified using a micro-BCA colorimetric assay (Pierce, Thermo Fischer Scientific, Dreieich, Germany). Protein samples were separated by SDS-polyacrylamide gel electrophoresis on 15% gels and subsequently transferred onto Immobilon^®-P PVDF membrane (Millipore, Bedford, MA, USA). Incubation of rabbit-anti-TSPO (ab109497), mouse-anti-ATPB (ab14730), and mouse-anti-VDAC1 (ab186321) antibodies, all from Abcam, Cambridge, UK, diluted were performed O.N. at 4 °C.

Whole cell protein samples were sonicated and boiled in RIPA buffer, and total protein was quantified using a micro-BCA colorimetric assay (Pierce, Thermo Fischer Scientific, Darmstadt, Germany). Protein samples were separated by SDS-polyacrylamide gel electrophoresis on 15% gels and subsequently transferred onto a nitrocellulose membrane (Amersham™ Protran 0.45 NC, GE Healthcare Life Sciences, Chicago, IL, USA). TSPO and VDAC1 proteins were detected using rabbit anti-TSPO (ab109497) and mouse anti-VDAC1 (ab186321) antibodies, both from Abcam, Cambridge, UK, respectively. Beta-1 tubulin antibody (clone E7, Developmental Studies Hybridoma Bank, Iowa, IA, USA) was used as a loading control. VDAC1 band densities were quantified using ImageJ2 Version 2.9.2. [30 (link)] and normalized to beta-1 tubulin.

Bax^−/−Bak^−/− MEFs and those reconstituted with the indicated BAX proteins were cultured as described above and 2 × 10⁷ cells were harvested for fractionation. Cell pellets were resuspended in isolation kit buffers (ThermoFisher) and lysed by Dounce homogenization. Mitochondrial and cytosolic fractions were purified by differential centrifugation, performed in accordance with the manufacturer’s protocol. Mitochondria were lysed in mitochondrial lysis buffer (25 mM Tris, 150 mM NaCl, 2% CHAPS, pH 7.2) and protein quantitation of the supernatant and cytosolic fractions was performed using the BCA assay (ThermoFisher). Samples were subjected to electrophoresis and western blotting using the 2D2 antibody (Santa Cruz Biotechnology Cat# sc-20067; RRID: AB_626726; 1:200). Western analysis using VDAC1 (Abcam cat #ab186321; 1:1000), LDH (Abcam cat #ab47010, RRID:AB_1952042; 1:1000), and actin (Cell Signaling Technology Cat# 5125, RRID:AB_1903890; 1:1000) antibodies was performed to verify mitochondrial and cytosolic fractions, and equal protein loading, respectively.