Semidry electroblotter

Cells were seeded in a six-well plate at a density of 4–6 × 10⁴ cells/well and allowed to incubate for 24 h. Extract was added to each well and incubated for 48 h. Cells were harvested and lysed in RIPA buffer (GenDEPOT) with protease inhibitors (Xpert protease inhibitor cocktail solution, GenDEPOT) and phosphatase inhibitors (Xpert phosphatase inhibitor cocktail solution, GenDEPOT). Cell lysates were boiled in 5x sample buffer and separated by 10% SDS-PAGE. Proteins were transferred onto PVDF membranes (Millipore) using a semidry electroblotter (Peqlab, Germany). Membranes were blocked with 5% skim milk in TBS-T (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) and sequentially incubated overnight with primary antibodies at 4°C. Membranes underwent additional incubation at room temperature and were then probed with secondary antibody. Immunoreactive proteins were visualized using ECL reagents and developed with X-ray film. Antibodies and dilutions are as follows: p53, p21 (1 : 2000, Millipore), c-Jun N-terminal kinase (JNK, 1 : 500, Santa Cruz Biotechnology), Bax (1 : 1000, Cell Signaling), caspase-3, caspase-8, caspase-9 (1 : 1000, Cell Signaling), β-actin (1 : 5000, Sigma-Aldrich), anti-mouse IgG (H+L) horseradish peroxidase conjugate, and anti-rabbit IgG (H+L) horseradish peroxidase conjugate (1 : 3000, Bio-Rad).

Cells were seeded in 6-well plate at a density of 4~6 × 10⁴ cells/well. Samples were treated to each well and incubated for 24 h or 48 h. Cells were harvested and lysed in RIPA buffer (GenDEPOT) with protease inhibitors (Xpert protease inhibitor cocktail solution, GenDEPOT) and phosphatase inhibitors (Xpert phosphatase inhibitor cocktail solution, GenDEPOT). Cell lysates were boiled in 5× sample buffer and separated by 10% SDS-PAGE. Proteins were transferred onto PVDF membranes (Millipore) using a semidry electroblotter (Peqlab, Germany). Membranes were blocked with 5% skim milk in TBST (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) and incubated sequentially with primary antibodies at 4°C, overnight. After washing membranes with TBST, the membranes reincubated with secondary antibody at room temperature for 3 hr. Immunoreactive protein was visualized using ECL reagents and developed with X-ray film. Antibodies and ratios at which they were used were p53, p21 (1 : 2000, Millipore), c-Jun N-terminal kinase (JNK, 1 : 500, Santa Cruz Biotechnology), β-actin (1 : 5000, Sigma-aldrich), and anti-mouse IgG (H+L) horseradish peroxidase conjugate and anti-rabbit IgG (H+L) horseradish peroxidase conjugate (1 : 3000, Bio-Rad).