Naringenin Modulates Cell Signaling Pathways

Following the previously established procedure^{24 (link)}, post 24 h naringenin treatment, the cells were lysed using RIPA buffer. Protein expressions of β-actin, MMP9, PI3K, AKT, and Caspase-3 were assessed through SDS-PAGE, transferred onto a PVDF membrane, the blots were cut prior to hybridization with antibodies, and cropped blots incubated overnight at 4 °C with primary antibodies including anti-MMP9 (Beyotime, 1:1000), anti-mTOR (Beyotime, 1:1000), anti-PI3K (Cell signaling, 1:1000), anti-p-PI3K (Cell signaling, 1:1000), anti-AKT (Cell signaling, 1:1000), anti-p-AKT (Cell signaling, 1:1000), anti-EGFR (ABclonal, 1:1000), anti-Caspase-3 (Beyotime, 1:1000), and anti-β-actin (Cell signaling, 1:1000). Secondary antibodies used were either GAM (goat anti-mouse IgG) or GAR (goat anti-rabbit IgG) (ABclonal, 1:2000).