2.8. Western Blot

Western blot analyses were performed to confirm the presence of differentially expressed proteins. After the treatment of the indicated concentration of ginsenoside F₂ (10, 20, and 40 μM) for 12 h, cells were harvested, washed with cold PBS (pH 7.4), and lysed with ice-cold lysis buffer (50 μM Tris-HCl, 150 μM NaCl, 1 μM EGTA, 1 μM EDTA, 20 μM NaF, 100 μM Na₃VO₄, 1%NP40, 1 μM PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin, pH 7.4) for 30 min and centrifuged at 12 000 ×g for 30 min at 4°C. The protein concentration of the clear supernatant was quantified using Bio-Rad Protein Assay Kit.

Approximately 30 μg of protein was loaded into a 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Thereafter, proteins were electrophoretically transferred to nitrocellulose membrane and nonspecific sites were blocked with 5% skimmed milk in 1% Tween-20 (Sigma-Aldrich) in 20 μM TBS (pH 7.5) and reacted with a primary polyclonal antibody, PRR5, CISD2, Bcl-2L, NLRX1, RPS15, RPL26, p53, Atg5, Atg7, Atg10, LC3-II, LC3-I PUMA, Beclin-1, UVRAG, and mTOR and β-actin for 4 h at room temperature. After washing with TBS three times (5 min each), the membrane was then incubated with alkaline phosphatase-conjugated goat anti-rabbit secondary antibody. The signal was observed and developed with Kodak film by exposure to enhanced chemiluminescence (ECL) plus western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ, USA).