Coxiv 3e11

The rabbit polyclonal CHCHD4 and rabbit polyclonal BNIP3 antibodies were purchased from Cambridge Biosciences. The mouse monoclonal HIF-1α antibody was purchased from BD Biosciences. The mouse monoclonal β-actin and rabbit polyclonal ATP5B antibodies were purchased from Abcam. The mouse monoclonal anti-myc (9B11) and rabbit monoclonal COXIV (3E11) antibodies were purchased from Cell Signaling Technology. The mouse monoclonal anti-Pimonidazole antibody was purchased from HypoxyProbe, Inc. The donkey anti-rabbit and anti-mouse horseradish peroxidase-linked secondary antibodies were purchased from VWR. Sodium azide was purchased from Sigma-Aldrich and used at 5 mM for 16 h in order to optimally block HIF-1α protein levels without significantly affecting cell viability. DAPI solution (10 mg/mL) was purchased from Cambridge Biosciences. Pimonidazole (200 mM) was purchased from HypoxyProbe, Inc.

Samples were resolved by SDS-PAGE (7.5–15% gels), transferred to Immobilon-P membranes (Millipore Inc.) and immunoblotted as indicated. Primary antibodies were used to detect Cox IV (3E11, Cell Signaling), cytochrome c (D18C7, Cell Signaling), Drp1 (BD Biosciences; D6C7, Cell Signaling; H-300, Santa Cruz biotechnology), Flag (Sigma), GAPDH (Abcam), GFP (FL, Santa Cruz biotechnology; Roche), GST (GE Healthcare), HA (Sigma), Mff (Proteintech; Sigma), RhoGDI (Abcam), SENP3 (D20A10, Cell Signaling), and β-actin (Sigma). Immune complexes were detected either using HRP-conjugated secondary antibodies (Sigma) followed by enhanced chemiluminescence (Thermo Scientific Pierce) or using fluorescent secondary antibodies (LI-COR). Each immunoblot presented is representative of at least three experiments carried out using different cell populations.

Reagents were purchased as follows: cisplatin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); etoposide and SP600125 from LC Laboratories (Woburn, MA, USA); SB203580, Roscovitine, LY294002, and CHIR99021 from Sigma-Aldrich; Cycloheximide and MG-132 from Millipore (Billerica, MA, USA); Phos-tag acrylamide from Wako Chemicals USA (Richmond, VA). PD184352 was kindly provided by Steven Grant (Virginia Commonwealth University), which was chemically synthesized in house based on the published structure of the drug. cisplatin was dissolved in PBS [30 (link)] and other reagents were dissolved in dimethyl sulfoxide. The following antibodies were used: Cleaved PARP (D64E10), Cleaved Caspase-3 (5A1E), BCL-X_L, BIM, phospho-S64 MCL-1, COX IV (3E11), β-Actin (13E5) and GAPDH from Cell Signaling Technology (Danvers, MA, USA); Noxa (114C307.1) from Thermo Fisher Scientific (Waltham, MA, USA); MCL-1 from Enzo Life Sciences (Farmingdale, NY, USA) and Rockland Immunochemicals (Gilbertville, PA, USA); BAK (06-536) from Millipore; BAX (N-20), alpha-Tubulin (sc-8035), Myc (9E10) and CDK1 (sc-54) from Santa Cruz Biotechnology; CDK2 from BD Biosciences; HA (3F10) from Roche (Indianapolis, IN, USA). [γ-³²P]ATP was purchased from PerkinElmer (Kanagawa, Japan). Histone H1, recombinant CDK2/Cyclin A, and CDK2/Cyclin E were purchased from SignalChem (Richmond, BC, Canada).