*2.6. Western Blot Analysis*

HCT116 cells treated with or without 10 ng/mL TNFα (Calbiochem, San Diego, CA, USA) in the presence or absence of compound **5** were lysed in a cell lysis buffer containing 20 mM HEPES (pH 7.2), 1% (*v*/*v*) Triton X-100, 10% (*v*/*v*) glycerol, 150 mM NaCl, 10 µg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein extracts (20 µg per sample) were separated via 10% (*w*/*v*) SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and incubated with the appropriate primary and secondary antibodies. Primary antibodies against phospho (p)-IKKα/β (Ser176/180), p-IκBα (Ser32), p-p65/RelA (Ser536) were obtained from Cell Signaling Technology (Beverly, MA, USA), and an antibody against glyceraldehyde phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All the primary antibodies were diluted to 1:1000 concentration in 50 mM Tris-buffered saline (pH 7.6) containing 5% nonfat dry milk solution and 0.05% Tween-20. The antibody-bound blots were developed using an enhanced chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA).
