Polycerasoidin-Induced Protein Expression

The western blotting assay of proteins extracted from the MCF7 cells treated with IC₅₀ concentration polycerasoidin was performed as previously described [24 (link)], with slight modifications. Briefly, protein separation (40 μg) was performed using 10% SDS-PAGE (20 mA, for 3 h). The separated proteins were transferred onto a PVDF membrane (Pierce, Rockford, IL, USA), which was subsequently blocked with Blocker Casein (Pierce) for 1 h at 25°C and then washed twice with TBST. The membranes were then incubated with the following primary antibodies at 4°C overnight: Bax (Cat: sc-493), Bcl-2 (Cat: sc-492), caspase-7 (Cat: sc-33773), caspase-9 (Cat: sc-7885), p21 (Cat: sc-397), p27 (Cat: sc-528) and β-actin (Cat: sc-7210) (1:1000; Santa Cruz Biotechnology, USA). This incubation was followed by a 1-h incubation with goat anti-rabbit and goat anti-mouse secondary antibodies conjugated to alkaline phosphatase (i-DNA, USA) at 25°C. After the membranes were washed twice with TBST, the bands were detected using the Fusion FX7 system (Vilber Lourmat, Germany).