Mouse monoclonal anti actin

Neuronal cells and tissue samples were directly lysed in boiling Laemmli buffer (60 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 5% beta-mercaptoethanol; 0.01% bromophenol blue). Nematodes were collected and resuspended in RIPA lysis buffer (Sigma-Aldrich). After sonication, equal amount of total proteins was separated by SDS polyacrylamide gel electrophoresis (12% to 15% percentage of acrylamide in the running gel) and transferred onto nitrocellulose membranes. Membranes were incubated for 1 h at room temperature (RT) with 5% non-fat milk in Tris-buffered saline, containing 0.05% Tween-20. Primary and secondary HRP-conjugated antibodies were incubated in the same buffer. Protein specific signals were detected using ECL Western Pico Detection system (ThermoFisher Scientific) and chemiluminescence signal visualized using Chemidoc imaging system (Biorad). The following primary antibodies and dilutions were used: guinea-pig polyclonal anti-p62 (1:1000; Progen), rabbit polyclonal anti-LC3B (1:1500; Sigma-Aldrich), mouse monoclonal anti-β-actin (1:5000; Sigma-Aldrich) and mouse monoclonal anti-actin (1:5000, Abcam). HRP-conjugated goat anti-mouse, anti-rabbit and anti guinea pig IgG (ThermoFisher Scientific, Waltham) were used as secondary antibodies.

Cells were treated with vehicle, quarfloxin, or CX-5461 as indicated. At harvesting, floating cells and adherent cells (trypsination) were collected for analysis. Protein isolation and blotting were performed essentially as previously described using NuPAGE 4–12% bis-tris precast polyacrylamide gels and Immobilon-PVDF membranes (Millipore) [42 (link)]. For western blots containing Caspase-3 and p21, 4–20% Tris-Glycine gels (Lonza) were used for better separation of these low MW proteins. Primary antibodies used in this study were mouse monoclonal anti-MycN (sc-53993, Santa Cruz Biotechnology, CA, USA), mouse monoclonal anti-p53 (sc-126, Santa Cruz Biotechnology, CA, USA), rabbit monoclonal anti-p21 (#2947, Cell Signaling Technology, MA, USA), rabbit polyclonal anti-PARP (#9542, Cell Signaling Technology, MA, USA), rabbit polyclonal anti-Caspase-3 (#9662, Cell Signaling Technology, MA, USA), mouse monoclonal anti-γ-H2A.X (05–636, Merck Millipore, MA, USA), rabbit polyclonal anti-actin (A2066, Sigma-Aldrich, MO, USA) and mouse monoclonal anti-actin (AB3280, Abcam, Cambridge, UK). Membranes were detected using the Odyssey Infrared Imaging system (LI-COR).