Cross-Linking Mast Cell Specific Gangliosides Stimulates the Release of Newly Formed Lipid Mediators and Newly Synthesized Cytokines

Figure 2

Cross-linking GD1b derived gangliosides by mAbAA4 increased cPLA₂ phosphorylation and induced COX-2 expression. Either RBL-2H3 mast cells were stimulated via FcεRI, by sensitizing the cells with IgE anti-TNP and stimulating with DNP₄₈-HSA (50 ng/mL), or cells were incubated with mAbAA4 (1, 2.5, 5, and 10 μg/mL) for 5 min (cPLA₂ phosphorylation) or for 1 h and then rinsed and cultured for an additional 3 h (COX-2 expression). Total cell lysates were immunoblotted with antibodies against p-cPLA₂, cPLA₂, COX-2, and α/β-tubulin (housekeeping protein) and the mean optical density of the bands was determined. Densitometry of the changes in expression and phosphorylation of proteins were corrected for α/β-tubulin. Data were expressed as the fold of nonstimulated (NS) cells. (a) Ratio of phosphorylated cPLA₂/total cPLA₂; (b) a representative blot from (a); (c) ratio of COX-2/α/β-tubulin; (d) a representative blot from (c). Data is expressed as the mean ± SD of three independent experiments. between experimental samples and the nonstimulated (NS) cells. between experimental samples and FcεRI stimulated cells.