Mitochondrial Protein Synthesis Assay

The assay was performed as described previously (6 (link)). Briefly, 2 × 10⁷ WT and CBFB_KO MCF10A cells were plated in complete DMEM/F12 media. The next day, cells were transferred to a methionine-free complete DMEM/F12 media with 5% dialyzed horse serum. The cytosolic translation was blocked by treating cells with cycloheximide for 20 minutes. For the negative control, cells were treated with both cycloheximide (50 μg/ml) and chloramphenicol (150 μg/ml) for 50 minutes to block mitochondria protein translation. Subsequently, cells were labeled with 500 μM L-Homopropargylglycine for 4 hours. After incubation, were extracted using the Mitochondria Isolation Kit for Cultured Cells (Abcam, Cat# ab110170) as per the manufacturer’s instructions. Next, 70 μg of mitochondria protein lysate was click-labeled with 40 μM AZDye 594 Azide using Click-&-Go^™ Protein Reaction Buffer Kit (Click Chemistry Tools, Cat#1262) as per the manufacturer’s instructions. Subsequently, lysates were run on a NuPAGE gel, and the gel was scanned at FLA-6500 Starion (Fujifilm) to visualize fluorescent proteins.