Immunoblotting of Mast Cell Signaling

Anti-DNP IgE (50 ng/ml)–sensitized mBMMCs were pre-treated with or without G.M2 for 1 h and then challenged with DNP-HSA (100 ng/ml) for 5 min (Lyn, Fyn, and Syk) and 15 min (PI3K, PLCγ1, Akt, IKK α/β, IκBα, and p65 NF-κB). Total protein was extracted as described previously (Kim et al., 2018 (link)). Equal amounts of protein were electrophoresed using 7.5–10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with specific primary antibody followed by anti-IgG horse-radish peroxidase-conjugated secondary antibody. Immunodetection was performed using an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ).