HCT116 cells were lysed in Triton X-100 lysis buffer [50 mM tris (pH 8.0), 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), protease inhibitors, and Benzonase]. After centrifugation at 13,000g for 10 min, the supernatants (1 mg of total protein) were collected and incubated with anti-Flag M2 affinity gel at 4°C for 2 hours with rotation. Samples were washed with lysis buffer four times and competed with 3×Flag peptides for 15 min with vigorous agitation. Proteins were resuspended in 5× SDS sample loading buffer, heated to 95°C for 5 min, and subjected to SDS–polyacrylamide gel electrophoresis (PAGE).