Monoclonal anti p62

Total cell extracts were prepared by incubation in lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 150 mM KCl, 1 mM dithiothreitol, 1 % Nonidet P-40) and a mix of protease inhibitors and resolved by 9–18 % SDS-polyacrilamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane (PVDF, Millipore) and membranes were blocked with 5 % nonfat dry milk in PBS and incubated with the following primary antibodies: monoclonal anti-poly (ADP-ribose) polymerase (PARP, BD Pharmingen, CA, USA), monoclonal anti-p53 (Ab-DO1), polyclonal anti-p53 (FL393), monoclonal anti-p62 (Santa Cruz Biotechnology, Dallas, TX, USA), monoclonal anti-phospho-Histone H2AX (Ser139) (Millipore, clone JBW301), and monoclonal anti-LC3B (Sigma-Aldrich). Equal lane loading was monitored by probing membranes with antibodies specific for β-actin (Calbiochem, San Diego, CA, USA). Primary antibodies were detected with appropriate horseradish peroxidase-labeled secondary antibodies (Bio-Rad, Hercules, CA, USA). Enzymatic signals were visualized using chemoluminescence (ECL Detection system, GE Healthcare, Milan, Italy).

Cell culture reagents were purchased from Invitrogen. Earle’s balanced salt solution (EBSS, E3024), bafilomycin A1 (B1793), 3-methyladenine (3-MA) (M9281) and wortmannin (W1628) were purchased from Sigma Aldrich. For the western blotting experiments, the following antibodies were used: polyclonal anti-rat QSOX1 [5] (link) diluted at 1∶7500, polyclonal anti-LC3 (Sigma, L8918) diluted at 1∶3000, monoclonal anti-p62 (Santa Cruz, sc-28359) diluted at 1∶1500, polyclonal anti-actin (Sigma, A5060) diluted at 1∶15000, polyclonal anti-rabbit (P.A.R.I.S, BI2407) diluted at 1∶10000 and polyclonal anti-mouse (P.A.R.I.S, BI24130) diluted at 1∶10000. For the immunofluorescence experiments, the following antibodies were used: monoclonal anti-p62 antibody (Santa Cruz, sc-28359) diluted at 1∶250, monoclonal anti-mouse lysosomal-associated membrane protein 1 (LAMP1) (Abcam, Ab25630) diluted at 1∶100, Alexa Fluor 555 goat anti-mouse (Life technologies, A-21422) diluted at 1∶800.