2.3. COX4 Isoform Overexpression

COX4i1 (CCDS10955.1) and COX4i2 (CCDS13187.1) isoforms of the human COX4 subunit full-length cDNAs including the 3′ end sequence encoding the FLAG tag (DYKDDDDK) were obtained as synthetized DNA strands with 5′ and 3′ overhangs containing restriction sites used for insertion into the pcDNA3.1⁺ mammalian expression vector (Thermo Fischer Scientific). COX4i1-FLAG or COX4i2-FLAG constructs were transfected into COX4i1/2 KO cells using Metafectene Pro (Biontex Laboratories GmbH); the selection of stably-transfected cells was started 48 h post-transfection with 1 mg/mLG418. Single-cell clones were later established from G418-resistant cells, and three clones of each COX4i1 and COX4i2 knock-in cells were used for the following experiments.