Anti coxiv

Total intracellular protein was extracted using precooled RIPA lysis buffer. Protein concentrations were detected using a BCA protein assay kit (Beyotime). Samples with equal protein concentrations were separated by 10% SDS‐PAGE. The proteins were transferred to PVDF membranes (Roche). The membranes were blocked with skim milk for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. Membranes were incubated with appropriate HRP‐conjugated secondary antibody (ABclonal; 1:10 000) for 1 h at room temperature. Signals were detected using enhanced chemiluminescence reagent (ECL, Advansta). The following primary antibodies were used for Western blot analysis at the indicated dilutions: anti‐Mfn2 (Proteintech, 1:1000), anti‐Vdac (ABclonal, 1:1000), anti‐COXIV (ABclonal, 1:1000), anti‐Tom20 (ABclonal, 1:1000), anti‐Hif‐1α (ABclonal, 1:1000), and anti‐β‐actin (ABclonal, 1:1000).

We isolated mitochondrial and cytoplasmic proteins from transgenic GCO and 239 T cells transfected with pCS2 + -Drfundc1-Cherry-His and/or pCS2 + -Cherry, strictly following the mitochondria protein extraction kit manual (Jiancheng Bioengineering Institute, Nanjing, China; G006). DrFundc1, Cherry, FUNDC1, COX IV, LC3B, and β-ACTIN were detected by use of Western blotting. Antibodies used were anti-His (Beyotime Biotechnology, Shanghai, China; AH367), anti-Cherry (ABclonal, Wuhan, China; AE002), anti-FUNDC1 (Bioss Inc., Boston, MA, USA; bs-13227R), anti-COX IV (ABclonal; A10098), anti-LC3B (ABclonal; A7198), and anti-β-ACTIN (Bioss Inc.; bsm-33036M).