ART-probe Labeling in Cell Lysates

Cells were cultured in six-well plates until 80–90% confluence was reached. As described before [20] (link), ART-probe (20 μM) in 2 ml of medium with a final DMSO concentration of 1% was added, and the cells were incubated at 37 °C with 5% CO₂ for 6 h. After treatment, the cells were lysed to obtain total cell lysates or harvested to isolate mitochondrial fraction according to the manufacture (Thermo Fisher Scientific, 89874). Equal amounts of the extracted proteins were then subjected to fluorescence labeling. The “click” reaction was done by adding Rhodamine B-azide (10 μM), TCEP (1 mM), TBTA (100 μM), and CuSO₄ (1 mM) to the lysate, followed by 2 h incubation at room temperature. The labeled proteins were then acetone-precipitated and air-dried. The samples were then solubilized with 100 µL of 1× SDS loading buffer. Sample was separated with 4–20% gradient SDS-PAGE gel. Typhoon 9410 laser scanner (GE Healthcare) was used to obtain the gel images, which were analyzed by Image Quant software.