Clarity ecl substrate solution

The lysates of cells were prepared in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in 1X PBS, pH 8.0) with a protease inhibitor cocktail (Roche, Switzerland). Protein concentrations were determined via BCA assay (Thermo Fisher Scientific, US). At first, equal amounts of prepared were denatured with 4X Laemmli buffer at 95 °C. Then denatured samples were loaded into gels and separated by SDS-PAGE electrophoresis. After electrophoresis was completed, gels were transferred to PVDF membranes (EMD Millipore, US, IPVH00010). After blocking membranes in 5% non-fat dry milk prepared in 1X PBS-0.1% Tween-20, membranes were labeled with primary and secondary antibodies. In this study, antibodies against hTERT (Origene, TA301588) and GAPDH (CST, 5174) were used. Secondary antibodies (Goat anti-rabbit-31460 and Goat anti-mouse-31430) were used. The proteins’ chemiluminescence signals were determined with Clarity ECL substrate solution (BIORAD, US, 1705061) by Vilber Loumart FX-7 (Thermo Fisher Scientific, US). ImageJ software (http://imagej.nih.gov/ij/) was used for Western blot images.

Cell lysates were prepared by RIPA buffer (1XPBS, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, US). After equal amounts of proteins were loaded to the gels, proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (EMD Millipore, Thermo Fisher Scientific, US). Following the classic immunoblotting steps (blocking, incubating with primary and secondary antibodies), chemiluminescence signals were detected using Clarity ECL substrate solution (Bio-Rad, US) by Fusion-FX7 (Vilber Lourmat, Thermo Fisher Scientific, US). Monoclonal antibodies used in this study were anti-LC3 (CST-12741, USA), anti-Atg-5/12 (CST-12994, USA), anti-Atg-7 (CST-8558, US), anti-actin (Sigma-Aldrich-A5316, UK), anti-caspase-3 (CST-9665, US), Anti-CHOP (CST-2895, UK). The experiments were repeated three times independently; with one representative result shown.