Journal: Molecular and Cellular Biology
Article Title: C11orf83, a Mitochondrial Cardiolipin-Binding Protein Involved in bc 1 Complex Assembly and Supercomplex Stabilization
Figure Lengend Snippet: C11orf83, like OPA1, is cleaved by OMA1 under ΔΨ loss. (A) Analysis of the stress-induced proteolysis of C11orf83 compared to OPA1. HeLa cells expressing C11orf83-V5 were treated with mitochondrion-damaging agents for 3 h, and cell lysates were analyzed by immunoblotting after SDS-PAGE. β-Catenin and SDHB were used as loading controls for cytosolic and mitochondrial proteins. Stress-induced cleavage of OPA1 and C11orf83 was observed by antibodies against OPA1 and the V5 tag. ROS, reactive oxygen species. (B) Analysis of the sensitivity of CCCP-induced cleavage of C11orf83 to various protease inhibitors. HeLa cells expressing C11orf83-V5 were incubated with CCCP with or without protease inhibitors. Cells lysates were fractionated by SDS-PAGE and analyzed by immunoblotting. β-Catenin and SDHB were used as loading controls. The prevention of C11orf83-V5 proteolysis was monitored thanks to an antibody against the V5 tag. (C) OMA1 involvement in the stress-induced cleavage of C11orf83. HeLa cells were transfected with C11orf83-V5 and treated with control siRNA or siRNA against OMA1. Forty-eight hours after siRNA transfection, cells were incubated with or without CCCP and cell lysates were analyzed by immunoblotting, after SDS-PAGE, using antibodies against OMA1, OPA1, and the V5 tag. GAPDH was used as a loading control. The two bands detected by the antibody against OPA1 in the absence of CCCP correspond to short and long OPA1 isoforms. The degradation of endogenous C11orf83 under CCCP treatment (lane 3) was prevented by the depletion of OMA1 (lane 4).
Article Snippet: Rabbit polyclonal antibodies obtained from commercial sources were anti-FIS1 (sc-98900) and anti-SIRT3 (sc-99143) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-COX4 from Cell Signaling Technology (Danvers, MA), anti-OMA1 (NBP1-56970) from Novus Biological (Littleton, CO), anti-β-catenin (C2206) from Sigma-Aldrich, and anti-DIABLO (ADI-905-244) from Enzo Life Sciences.
Techniques: Expressing, Western Blot, SDS Page, Incubation, Transfection