Western blot analysis

Protein expression of hTERT was examined by Western blot. MCF-7 cells were seeded in 25 cm² flasks at a density of 1.5×10⁶ cells. Cells were treated for 48 hours with or without various concentrations of ENL and END (1, 10, 100 µM) and 10 nM of E2 as the positive control, then washed with phosphate-buffered saline (PBS) and lysed with RIPA lysis buffer that contained a mixture of protease inhibitors. Following quantification of total protein, 50 µg of the whole cell lysate was denatured in Laemmli buffer at 95˚C for 5 minutes, resolved on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in tris-buffered saline (TBS) that contained 0.05% Tween 20 and 3% bovine serum albumin (BSA) for 1 hour at room temperature. Blots were then incubated with rabbit anti-hTERT antibody (Novus Biologicals, USA) and rabbit anti-β-actin antibody (Cell Signaling Technology, USA) overnight at 4˚C. For visualization, blots were incubated with horseradish peroxidase (HRP)- conjugated secondary anti-rabbit antibody (Cell Signaling Technology, USA) for 1 hour. Membrane-bound secondary antibodies were visualized with a chemiluminescence HRP substrate (Roche Applied Sciences, Germany). Densitometry analysis was performed using ImageJ 1.17 (NIH, USA) to determine relative amounts of protein.