Rabbit anti mouse β actin

Samples were denatured in 50 mM Tris-HCl (pH 6.8) (Invitrogen), 100 mM dithiothreitol (DTT) (Sigma), 2% SDS (Invitrogen), 0.1% bromophenol blue (Sigma), and 10% glycerol (National Diagnostics) at 95°C for 5 min. Samples were run on 10% SDS-PAGE gels, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked with ~5% skim milk in PBS with 0.1% polysorbate 20 (PBST). Primary and secondary antibody incubations were for 18 h at 4°C and 1 to 2 h at room temperature, respectively. For the modified Western blot, murine and human TG homogenates were used as the primary antibody. TG homogenate was created from perfused tissue that was homogenized with a tissue blender (Omni International) in PBS plus protease inhibitor (Roche) as described above. Binding of TG IgG to the blot was visualized using an appropriate secondary antibody. Recombinant viral glycoproteins gH/L, gB, gC, and gD and polyclonal antibodies as positive controls were generously provided by Roselyn J. Eisenberg and Gary H. Cohen (University of Pennsylvania [30 (link)]). The antibodies and dilutions used were rabbit anti-mouse β-actin (BioLegend) at 1:1,000, goat anti-rabbit IgG (H+L) conjugated to horseradish peroxidase (HRP) (BioRad) at ~1:35,000, goat anti-mouse IgG (H+L) conjugated to HRP (Biorad) at ~1:35,000, and goat anti-human IgG (H+L) conjugated to HRP (Thermo Fisher Scientific) at ~1:35,000.