Anti parp1

For γH2AX foci detection, cells were fixed with 4% paraformaldehyde and subsequently with methanol for 10 min at −20 °C. After permeabilization and blocking, the slides were incubated with anti-γ-H2AX (Cell Signalling Technology Danvers, MA, USA) and Alexa594-conjugated secondary antibody with 4′,6-diamidino-2-phenylindole (DAPI) staining. For immunoblot analysis, cells were lysed with Laemmli’s buffer and then sonicated, and protein concentration was measured by a Protein Assay (Bio-Rad, Hercules, CA, USA). Proteins were then subjected to electrophoresis on an SDS-polyacrylamide gel followed by transfer to a Sequi-Blot^TM PVDF membrane (Bio-Rad) as described elsewhere. Immunoblot analysis was carried out using the following primary antibodies: anti-γ-H2AX (1:1000; Millipore, Burlington, MA, USA), anti-PARP-1 (1:1000; Cell Signalling Technology), and anti-β-actin (1:50,000, Sigma-Aldrich, St. Louis, MI, USA). The secondary antibodies were horseradish peroxidase-linked immunoglobulin, and immune complexes were detected using an enhanced chemiluminescence reaction kit (Millipore).

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.