HCT116 cells were transfected with flag-pcDNA3.1 plasmids or flag-MST3 plasmids. Whole-cell extracts were prepared with a lysis buffer, lysed on ice for 10 min, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The anti-Flag antibody and protein A/G beads were added and incubated for 6-8 h and washed five times with wash buffer. Proteins were eluted with 0.15 M glycine (pH 2.5-3.1), and the supernatant was collected and immediately added to 1/10 volume of neutralization buffer (0.1 M NaOH) to adjust the pH of the eluted product to neutral. Mass spectrometry analysis was performed.