ART3 Protein, Human (HEK293, His)

Materials
Cell line used for assay: MDA-MB-231
Cell culture medium: DMEM +10% FBS+1% P/S (DMEM complete medium)
Penicillin-Streptomycin (100×), Sterile (HY-K1006)
Cell Counting Kit-8 (CCK8 solution, HY-K0301)
ART3 Protein, Human (HEK293, His) (HY-P74409)

Procedure
1. Seed cells at a density of 3000 cells/well in a 96-well plate, with a total volume of 100 μL/well. (Do not seed cells in the outermost ring of the 96-well plate; instead, add an equal volume of PBS solution.)
2. Add 100 μL of DMEM complete mediumcontaining different concentrations of ART3 protein to the 96-well plate, achieving final concentrations of ART3 protein in each well of 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 25, 50 μg/mL.
3. Incubate the cells in a 37°C incubator with 5% CO₂ for 72 hours.
4. After the incubation period, add 10 μL of CCK-8 solution to each well under light-protected conditions and incubate in a 37°C incubator with 5% CO₂ for 2 hours.
5. Measure the OD value at 450 nm using a microplate reader.
6. Using GraphPad Prism software, plot a curve with the OD value as the vertical axis and the protein concentration as the horizontal axis, and calculate the ED₅₀ value.