Mouse anti ha antibody

The crude cell extracts were prepared in a buffer containing 200 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10% glycerol (v/v), separated by SDS-PAGE, and blotted to nitrocellulose. The immuno-detection of proteins was carried out using mouse anti-HA antibody (#26183, ThermoFisher Sci) or mouse anti-LexA (#306–719, EMD Millipore Corp). The secondary antibodies used were anti-mouse IgG antibodies conjugated with horseradish peroxidase (#A9044, Sigma Aldrich). The proteins were visualized using Pierce ECL (#32106, ThermoFisher Sci) according to the manufacturer’s instructions.

HEK293T cells were transfected with pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector, collected at 36 hpt, fixed with 4% paraformaldehyde overnight at 4 °C, and permeabilized with 0.25% Triton X-100 for 30 min at 4 °C. The cells were rinsed three times with PBST (containing 0.1% Tween-20), blocked with 5% BSA PBS for 2 h at 37 °C, and then incubated with a mouse anti-HA antibody (MBL, Japan, 1:100), goat anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, and Alexa Fluor 488 (Thermo Fisher Scientific, Meridian Road Rockford, USA, 1:1000). All antibodies were diluted in 1% BSA PBS. Finally, cell nuclei were visualized with DAPI (Roche, Mannheim, Germany). Coverslips were sealed with glycerin buffer, and the cells were visualized using a fluorescence microscope (Nikon ECLIPSE 80i, Japan) [46 (link)].