Hrp anti mouse secondary antibody

AMs (3×10⁵·well⁻¹) were infected with RSV (MOI 1) for the indicated times. Cells were then harvested and lysed in RIPA buffer containing a protease inhibitor cocktail. Cell lysates were boiled and subjected to electrophoresis in SDS-polyacrylamide gel (10%) in reducing conditions. The quantified proteins were transferred to a methanol-activated PVDF membrane at 4°C for 2 h. Then, the membranes were blocked with non-fat milk-TBS-T solution for 1 h at room temperature and incubated overnight with anti-MLKL antibodies (1:1000; cat# AP14272, Abgent, San Diego, CA, USA) followed by horseradish peroxidase (HRP) anti-rabbit secondary antibody (1:5000; cat#31460, ThermoFisher Scientific). As an endogenous control, anti-GAPDH antibody (1:5000; cat# MA5-15738, ThermoFisher Scientific), followed by HRP anti-mouse secondary antibody (1:5000; cat# 31430, ThermoFisher Scientific) was used. MLKL expression was detected using the ECL system (Amersham ECL Prime Western Blotting Detection reagent; GE Healthcare Life Sciences, Chicago, IL USA) and the membrane was imaged using the ChemiDoc Imaging System (BioRad, Hercules, CA, USA). Densitometry analysis was performed using ImageJ 1.43 software (US National Institutes of Health, Bethesda, MD, USA). MLKL bands were normalised to GAPDH bands.