Quantifying Cell Cycle Protein Levels

Identification of the level change of cell cycle proteins (CDK2 and Cyclin D1) was investigated in this study. The effect of HAA₂₀₂₀ (500, 2500, 5000 nM), dinaciclib (5, 25, 50 nM), and their combination in MCF7 cells was tested following a previous report [23 (link)]. Briefly, MCF7 cells (1 × 10⁶ cells/well of a 6-well plate) were treated for 24 h. The lysis buffer was used to isolate total proteins, while the Bradford method was used to determine their concentration, which were then electrophoresed using a polyacrylamide gel and transferred to membrane. The membranes were incubated with CDK2 (# 2561, 1:1000, Cell signaling) and Cyclin D1 antibodies (# 2922, 1:1000, Cell signaling) for 2 h at room temperature and secondary antibody GAPDH (# 8884, 1:1000, Cell signaling) for 1 h. Visualization of the immunoreactivity was assessed using horseradish peroxidase (HRP)-conjugated secondary antibodies.