Anti cypd

Catalog numbers and dilution fold are listed in parentheses. The following antibodies were purchased from Cell Signaling Technology: Anti-Bak (3792; 1:1000), anti-Bax (2772; 1:1000), anti-Bcl-2 (2870; 1:1000), anti-Bcl-xL (2764; 1:500), anti-CHK1 (2360; 1:1000), anti-phospho-CHK1 (S345; 2348; 1:1000), anti-cleaved caspase-3 (9661; 1:1000), anti-cleaved caspase-7 (9491; 1:1000), anti-cleaved caspase-8 (9496; 1:500), anti-cleaved caspase-9 (9501; 1:1000), anti-cytochrome c (4272; 1:500), anti-H2A.X (7631; 1:1000), anti-phospho-H2A.X (S139; 9718; 1:1000), anti-p53 (2524; 1:1000), anti-p53 (2527; 1:1000), anti-phospho-p53 (S6; 9285; 1:1000), anti-phospho-p53 (S15; 9284; 1:1000), anti-p21 (2946; 1:1000), and anti-voltage-dependent anion channel (VDAC; 4866; 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology: Anti-CD95 (1023; 1:500), anti-cyclophilin 40, also known as anti-CYPD (137157; 1:400), anti-His tag (803; 1:500), anti-lamin A (20680; 1:1000), anti-MDM2 (965; 1:500), and anti-ubiquitin (8017; 1:200). Anti-PEPD (Ab86507; 1:500) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; MAB374; 1:5000) were purchased from Abcam and EMD Millipore, respectively. Anti-mouse IgG-horseradish peroxidase (IgG-HRP; NA931V; 1:4000–5000) and anti-rabbit IgG-HRP (NA934V; 1:5000) were purchased from GE Healthcare.

After treatment, 600 μg of cell lysates from mitochondrial fractions of glioma cells were pre-cleared. The supernatant was then rotated overnight with 2 μg of anti-Cyp-D (Santa Cruz Biotech). Next, the lysates were centrifuged for 5 min at 4 °C in a micro-centrifuge to remove nonspecific aggregates. The protein A/G PLUS-agarose (35 μl, Sigma) was then added to the supernatants for 4 h at 4 °C. Pellets were washed six times with PBS, resuspended in lysis buffer, and then assayed by Western blots. The time point for IP was based on previous publications and results from pre-experiments.

Samples were homogenized in ice‐cold ‘immunoprecipitation buffer’ containing 20 mmol/L Tris‐HCl, pH 7.5, 150 mmol/L NaCl, and 1.5% Nonidet P‐40 supplemented with protease inhibitors. The extracts (500 µg protein per sample) were pre‐cleared with protein A/G beads and then mixed with non‐specific IgG (1 µg) or polyclonal mouse anti‐Bcl2 antibody (Cell Signaling Biotechnology), or anti‐CypD (Santa Cruz Biotechnology) overnight at 4°C, followed by the addition of 40 µL of protein A/G‐agarose (Santa Cruz Biotechnology) for 3 hours at 4°C. Immune complexes were washed four times in an ‘immunoprecipitation wash buffer’ (100 mmol/L Tris‐HCl, pH 7.5, 100 mmol/L NaCl, 0.1% Triton X‐100) and resuspended in 2× Laemmli buffer (Santa Cruz Biotechnology). The inputs were also mixed with 2× Laemmli buffer, and then the immunoprecipitation reactions and inputs were boiled for 10 minutes and pelleted in a centrifuge. The supernatants were subjected to Western blot analysis as described above.