Novex ecl hrp chemiluminescent detection system

Twenty-five micrograms of total protein extracts prepared according to standard methods were fractioned by SDS-PAGE (4–12% Bis–Tris/SDS polyacrylamide gel, NuPAGE, Thermo Fisher Scientific) and transferred onto Hybond nitrocellulose filters (GE Healthcare Life Sciences, Buckinghamshire, UK). Filters were blocked for 1 h at room temperature in 1× PBS-Tween20, 5% skim milk, and then incubated overnight at 4 °C with primary antibodies: Rabbit polyclonal anti-γ-H2AX (ab11174, Abcam, Cambridge, UK); anti-LAMP-1 (ab24170, Abcam); anti-LC3B (ab51520, Abcam); and anti-PARP-1 (#9542, Cell Signaling Technology, Danvers, MA, USA). Mouse monoclonal anti-GAPDH (G8796, Sigma-Aldrich S.r.l.) and anti-Vinculin (VCL, V9131, Sigma-Aldrich S.r.l.) antibodies were used to ensure equal protein loading. The filters were then probed with secondary, peroxidase-linked whole antibodies (GE Healthcare) for 1 h at room temperature, and blotted proteins were detected using the Novex^® ECL HRP Chemiluminescent detection system (Thermo Fisher). Filters were then subjected to autoradiography, the films were scanned, and images were analyzed using ImageJ 1.46r.

Forty micrograms of total protein extracts prepared according to standard methods was fractioned by SDS-PAGE, transferred onto nitrocellulose filters and incubated o.n. with primary antibodies: mouse monoclonal antibodies raised against β–actin, PAX8, TNKS, WRAP53; rabbit polyclonal antibodies raised against H2AX, γ- H2AX (S139), p21^Waf1, POT1, PRKCA (Y124), TEP1 and a goat polyclonal antibody raised against TNKS2 (Abcam, Cambridge, UK); rabbit polyclonal antibodies raised against TERF2, TSPYL5 and the mouse monoclonal raised against p53 (Santa Cruz Biotechnology); rabbit polyclonal antibodies raised against PARP1 and CPP32 (Cell Signaling Technology, Danvers, MA, USA). The filters were then probed with secondary peroxidase-linked whole antibodies (GE Healthcare, Milano, Italy) and detected by Novex® ECL HRP Chemiluminescent detection system (Thermo Fisher). Filters were then subjected to autoradiography. β-Actin (ACTB) was used on each blot to ensure equal protein loading.