Expressplus 10 8

Tenocytes were lysed, and 15 µg of each sample was separated on a 4–20% SDS-PAGE Gel by electrophoresis (ExpressPlus. 10 × 8, GenScript Biotech Corporation, Nanjing, China). After that, samples were transferred to nitrocellulose membranes, as already described [22 (link)]. Next, membranes were incubated in the presence of mouse monoclonal anti-β-actin (1:10,000) (Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-iNOS (1:200), rabbit polyclonal anti-Nrf2 (1:750) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit monoclonal anti-h-TERT (1:500) (ThermoFisher Scientific, Waltham, MA, USA). After an overnight incubation at 4 °C with primary antibodies under gentle shaking, membranes were then probed with specific IgG horseradish peroxidase (HRP)-conjugated secondary antibodies and bands were identified by chemiluminescence as previously described [22 (link)]. At least three independent experiments were performed for each protein. Results are expressed as mean values ± standard deviation (S.D.) of normalized densitometric values on the loading control (β-actin).

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)].