*3.8. Western Blotting*

Western blot analysis was performed in triplicate as previously described [14]. Cells were treated with compound PN (0.5–4 μg/mL) for 48 h and detached with trypsin-EDTA. Proteins were isolated with RIPA buffer containing protease inhibitors (Sigma) and quantified using a bicinchoninic acid (BCA) assay (Thermo Scientific). Proteins were separated in precast SDS-PAGE gels (10%, Invitrogen) and transferred to membranes using the iBlot system (Thermo Scientific). Membranes were blocked with 5% skimmed milk in Tris buffered saline (TBS) and incubated with the following primary antibodies overnight at 4 ◦C; cdc2, cyclin B1, HSP90, phospho-mTOR, GAPDH, KRAS, phospho-ERK1/2, and phospho-AKT. Membranes were then washed and incubated with HRP-conjugated goa<sup>t</sup> anti-rabbit or anti-mouse antibodies (Pierce, Rockford, IL, USA). Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD, USA) and ratios of proteins versus the loading control were calculated.
