Western Blot Analysis of Apoptotic and Signaling Pathways in CRC Cells

Human CRC HT-29 and HCT116 cells were treated or not with the determined IC₅₀ values of compounds (12a, 10a, 10b) for indicated times (6, 12, 24 and 48h) and then harvested with trypsin. For total protein extraction, collected samples of each condition were washed with PBS. Then, the total cell pool was centrifuged at 200× g for 5 min at 4 °C and homogenized in RIPA lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.1% Sodium Dodecyl Sulfate (SDS), 20 mg/mL of aprotinin) containing protease inhibitors according to the manufacturer’s instructions as previously described [30 (link)]. Proteins (60 µg) were separated on 12.5% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with respective human antibodies against caspase-3, cleaved caspase-3, PARP and Akt, ERK, p38 MAP Kinases and its phosphorylated forms according to the manufacturer’s instructions. After incubation with appropriate secondary antibodies, blots were developed using the «Immobilon Western » substrate following the manufacturer’s protocol and G:BOX system (Syngene, Ozyme). Membranes were then reblotted with human anti-β-actin used as a loading control.