Typhoon 9410 laser scanner

HCT116 cells were cultured in six-well plates until 80 – 90% confluence was reached. Then the cells were washed thrice with PBS. Cur-P (5–60 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 4 h. Control treatment was done with the same volume of medium containing 1% DMSO. After treatment, the cells were washed with PBS and trypsinized to detach from the plate. The cells were pelleted, resuspended in PBS, washed, and subjected to sonication in 100 μl of PBS to lyse the cells. Centrifugation (10,000 rpm; 45 min) was applied to remove the insoluble fraction from the cell lysate. Bradford assay was used to determine the protein concentrations of the supernatants. Equal amounts (50 μg) 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. Fifty microliter of 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.

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.

In parallel wells, cells were cultivated after transfection for 48 h, collected, suspended in 200 µl 0.1 M Tris.HCl buffer, pH 7.6 containing 0.25 M sucrose, and protein content was determined by the Bradford assay with serum albumin as standard. 80 µg protein were then separated on 12.5% SDS gels, blotted to PVDF membranes (GE Healthcare, Vienna, Austria), incubated with a rabbit polyclonal anti-myc antibody (abcam ab9106), washed, incubated with a goat-anti-rabbit Cy5 labeled antibody (Fisher Scientific, Vienna, Austria), and the amount of fluorescence detected with a Typhoon 9410 laser scanner (GE Healthcare, excitation 630 nm, emission 670 nm).