Ab130561

The following antibodies were used in this study at the indicated dilution for western blot (WB) analysis, IP, IHC, and immunofluorescence (IF): Nedd4 (PA5-17463, Thermo, 1:1000 for WB, 1:100 for IP, 1:100 for IHC), VDAC1 (MABN504, Merck, 1:1000 for WB), VDAC2 (ab37985, Abcam, 1:1000 for WB), VDAC3 (ab130561, Abcam, 1:1000 for WB, 1:100 for IHC), FOXM1 (702664, Thermo, 1:1000 for WB), p-ERK (4370, Cell Signaling, 1:1000 for WB), ERK (4695, Cell Signaling, 1:1000 for WB), 4HNE (ab46545, Abcam, 1:100 for IHC), Flag (F3165, clone M2; Sigma, 1:1000 for WB, 1:100 for IP), HA (H6533, Sigma, 1:1000 for WB), GST (PA1982A, Thermo, 1:1000 for WB), Myc (AH00052, Thermo, 1:1000 for WB, 1:100 for IP), and Actin (PA116889, Thermo, 1:1000 for WB). Horseradish peroxidase (HRP)-labeled or fluorescently labeled secondary antibody conjugates were purchased from Molecular Probes (Thermo). Purified rabbit IgG was purchased from Pierce. Erastin (E7781), Ferrostatin-1 (S7243), Liproxstatin-1 (S7699), EUK134 (S4261), and Tempol (S2910) were obtained from Selleck (Houston, TX, USA). DFO (D9533) and CPX (SML2011) were obtained from Sigma (St. Louis, MO).

Commercially available primary antibodies were used: rabbit polyclonal antibodies against CypD (239784, Calbiochem), PKCε (sc-214, Santa Cruz), actin (sc-1615, Santa Cruz), VDAC2 (ab47104, Abcam) and VDAC3 (ab130561, Abcam), and a mouse monoclonal antibody against VDAC1 (ab14734, Abcam). Cells were washed 3 times in PBS in 48-well plates and each well on a heat block was treated with 10 μl of boiled sample buffer (2% SDS, 1% NP40, 5% sucrose in 62 mM Tris–HCl, pH 6.8) containing protease inhibitors (50 μM PMSF, 50 μM leupeptin, 1 μg/ml aprotinin, and 0.5 μM pepstatin). After scraping, the recovered cells were further disrupted by sonication at output 10 for 30 s with a TOMY ultrasonic disruptor UD-200 and centrifuged at 20,000 × g for 3 min at 4 °C. The supernatants, supplemented with 2-mercaptoethanol and bromophenol blue, were again boiled and were loaded into a 12% (for CypD), 10% (for VDAC) or 8% (for PKCε) sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred to a PVDF membrane. Membranes were blocked with 5% non-fat milk for 2 h in PBS, and then incubated for 1.5−2 days at room temperature with specific primary antibodies: After washing with 0.1% tween 20 in PBS, blots were incubated overnight with biotinylated secondary antibodies and detected using the ABC method (PK-6100, Vector Labs).