Cells were seeded (0.3 × 105/well) in a 6-well tissue culture-treated plate (Falcon®, Corning Incorporated, NY, USA) and left to adhere for 24 h. Next, MCF7 cells were exposed to compounds at the concentration of 3 µM for 24 h. Protein concentration was determined using a bicinchoninic acid assay (QuantiPro™ BCA Assay kit for 0.5–30 μg/mL protein, Sigma-Aldrich, Milan, Italy) following the manufacturer’s instructions as already reported [46 (link)].
The MCF7 cell lysates (20 μg/sample) were electrophoresed on a 4–20% SDS-PAGE Gel (ExpressPlus™ 10 × 8, GenScript Biotech Corporation, Nanjing, China) and transferred to nitrocellulose membranes. Nitrocellulose membranes were afterward blocked in 5% of non-fat milk or 5% of BSA, 10 mmol/L Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20, and probed with the following primary antibodies: mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) (dilution 1:10,000), rabbit monoclonal anti-Akt, anti-phospho-Akt and anti-COX2 (all purchased by Cell Signaling Technology, MA, USA) (dilution 1:1000), goat polyclonal anti-caspase 3 (dilution 1:200), and rabbit polyclonal anti-Nrf-2 (dilution 1:750) (all purchased by Santa Cruz Biotechnology, CA, USA). Next, membranes were incubated and immunoreactive bands were detected as described previously [46 (link)].
The MCF7 cell lysates (20 μg/sample) were electrophoresed on a 4–20% SDS-PAGE Gel (ExpressPlus™ 10 × 8, GenScript Biotech Corporation, Nanjing, China) and transferred to nitrocellulose membranes. Nitrocellulose membranes were afterward blocked in 5% of non-fat milk or 5% of BSA, 10 mmol/L Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20, and probed with the following primary antibodies: mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA) (dilution 1:10,000), rabbit monoclonal anti-Akt, anti-phospho-Akt and anti-COX2 (all purchased by Cell Signaling Technology, MA, USA) (dilution 1:1000), goat polyclonal anti-caspase 3 (dilution 1:200), and rabbit polyclonal anti-Nrf-2 (dilution 1:750) (all purchased by Santa Cruz Biotechnology, CA, USA). Next, membranes were incubated and immunoreactive bands were detected as described previously [46 (link)].
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