4.5. Western Blot Analysis

DTC cells were treated with adavosertib (500 nmol/L) or vehicle for 24 and 48 h after plating overnight the cells at 1 × 10⁶ cells in 100-mm petri dishes in 10 mL of media. Cell pellets were dissolved using immunoprecipitation lysis buffer containing protein phosphatase inhibitor mixture (Bionovas, Toronto, ON, Canada), sonicated, and clarified by centrifugation. Equal amounts of protein lysate were separated by 12% Tris-HCl gels, transferred to polyvinylidene difluoride membranes, blocked with 5% fat-free milk, and exposed to the primary antibody followed by a secondary antibody conjugated to horseradish peroxidase. Proteins were detected by an enhanced chemiluminescence kit (PerkinElmer, Waltham, MA, USA) using UVP ChemStudio PLUS touch (Analytik Jena, Jena, Germany).

Band densitometry was performed using Molecular Imager VersaDoc MP 4000 system software (Bio-Rad, Hercules, CA, USA). The ratios of Wee1, PLK1, p-CDK1 (Tyr15), p-CHK1 (Ser345), AXL, cyclin E1, and Myt1 to β-actin were calculated in each cell line to determine the relative expression using untreated FTC-133 cell values as reference. Original western blot images could be viewed in File S1.