Chemoluminiscent reagent

Protein extracts were obtained by adding sample buffer to the cells, which were scraped and lysed by pipetting and boiling. Equal amounts of protein were loaded and separated in a SDS-PAGE; then, proteins were transferred onto a nitrocellulose membrane and incubated with the proper primary antibody. Membranes were blocked using 7.5% milk in TBS/T buffer. Primary antibodies were prepared in TBS/T buffer as follows: anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH, 1:1000) (Santa Cruz Biotechnologies), anti-SOD2 (1:1000) (Cell Signalling, Danvers, MA, USA), anti-SOD1 (1:1000) (Cell Signalling), anti-catalase (1:500) (Cell Signalling), anti-γH2AX (1:1000) (Abcam, Cambridge, UK), anti-H2AX (1:1000) (Upstate, Darmstadt, Germany), anti-His (1:1000) (Cell Signalling), anti-HA (1:1000) (Roche), anti-FLAG M2 (1:2000) (Sigma-Aldrich), and anti-HPV18 E2 (1:1000) (Abcam). Membranes were washed three times with PBS/T and incubated with HRP coupled secondary anti-mouse or rabbit antibodies (Santa Cruz Biotechnologies). Membranes were finally developed using the ChemoLuminiscent Reagent (Merck Millipore, Billerica, MA, USA) accordingly to the manufacturer's instructions. Densitometric analysis was performed using the ImageJ program ver.1.48h3, National Institutes of Health (NIH).