Mouse anti p62 ab56416

For western blot analysis, the total protein concentrations were determined by a BCA assay kit (Thermo Scientific Pierce, Rockford, IL, USA). Equal amounts of protein (30 µg) were separated by gel electrophoresis on gels with the indicated percentage of polyacrylamide, and transferred onto Immobilon-P membranes (Millipore, Etten-Leur, The Netherlands). The proteins were visualized using standard protocols. Primary antibodies rabbit anti-Atg5 (ab108327), mouse anti-LC3B (ab129376), rabbit anti-Atg3 (EPR4801) (Abcam, Cambridge, UK), mouse anti-p62 (ab56416) (Abcam, Cambridge, UK) were purchased from Abcam, rabbit anti-LC3B (NB600-1384) from Novus Biologicals (Littleton, CO, USA), mouse anti-βactin from MP Biomedicals (Santa Ana, CA, USA) and rabbit anti-Atg13 from Sigma-Aldrich (St. Louis, MO, USA). Primary 4F2 mouse anti-reovirus σ3 was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology (Iowa City, IA, USA) [20 (link)].

For immunostaining, cells were fixed with 4% paraformaldehyde in PBS, permeabilized and blocked with PBS containing 0.5% Triton X-100 and 3% BSA for 30 min at room temperature. The primary antibodies used were mouse anti-HA (sc-7392; Santa Cruz Biotechnology, Dallas, TX, USA, 1:200 dilution), mouse anti-p62 (ab56416; Abcam, Cambridge, UK, 1:500 dilution), rabbit anti-Tom20 (sc-11415; Santa Cruz Biotechnology, 1:200 dilution) and goat anti-hsp60 (sc-1052; Santa Cruz Biotechnology, 1:200 dilution). The secondary antibodies used were Alexa Fluor dye-conjugated donkey anti-mouse, donkey anti-goat and donkey anti-rabbit (Thermo Scientific, Waltham, MA, USA 1:200 dilution). Nuclei were counterstained with Hoechst 33342 (Thermo Scientific). All fluorescent images were acquired on Yokogawa CSU (confocal scanner unit)-W1 spinning disc system (Andor, Belfast, UK) equipped with a 60 × 1.49 NA objective (Nikon, Tokyo, Japan). The brightness of the images was adjusted using NIS-Elements (Nikon).