Murine monoclonal anti flag m2 antibody

After infection for the indicated times, J774A.1 lysates were pelleted by centrifugation at 13,400 × g for 15 min at 4°C and denatured at 95°C for 5 min in SDS-PAGE sample buffer containing 5% β-mercaptoethanol. Bacterial pellets or saponin extracts from equivalent cell numbers were separated by SDS-PAGE. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline plus 0.1% Tween 20 (TBST), incubated in primary and secondary antibodies diluted in 5% nonfat dried milk in TBST for 1 h each, and then washed with TBST. Primary antibodies (murine monoclonal anti-FLAG M2 antibody, Sigma-Aldrich catalog number F1804; goat anti-GFP, Rockland Immunochemicals [Limerick, PA] catalog number 600-101-215; and mouse monoclonal anti-NanoLuc antibody, Promega catalog number N7000) and secondary antibodies [goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, horseradish peroxidase (HRP), ThermoFisher catalog number 31432; and rabbit anti-Goat IgG (H+L) secondary antibody, HRP, ThermoFisher catalog number 31402] were used at a 1:1,000 or 1:5,000 dilutions, respectively. Western blot signals were visualized by chemiluminescence using the SuperSignal West Femto maximum sensitivity substrate (ThermoFisher Scientific) and a ChemiDoc system (Bio-Rad, Hercules, CA).