Gel doc image system

After transfected with mixture for 72 h, the HepG2 cells were collected and followed with Western blot analysis. The proteins were extracted with lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, sodium pyrophosphate, Na₃VO₄, 1 mM Phenylmethanesulfonyl fluoride (PMSF), and protease inhibitor cocktail). The samples (30 μg of protein) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membrane was incubated with anti-TERT rabbit monoclonal antibody [Y182] (1:1000, Sigma-Aldrich, St. Louis, MO, USA), β-actin (1:10,000, Sigma-Aldrich), then washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (GE Healthcare, Buckinghamshire, UK). The intensity of the bands was quantified using the Gel-Doc image system (Bio-Rad, Hercules, CA, USA).

NTD (WT and Y109A in 20 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5 droplet buffer) binding to the ss-14mer and ds-14mer was visualized by electrophoretic mobility shift assay in 2.5% agarose gels. Increasing concentrations of protein in the range of 0–125 μM were added to 25 μM RNA and incubated for 20 min at room temperature in a total reaction volume of 10 μL. About 2 microlitre loading dye was added to the reaction before loading. RNA bands were stained with Midori Green Nucleic Acid staining solution (Bulldog Bio. Inc., Portsmouth, NH) and visualized using a Bio-Rad Gel Doc Image system.