Effects of cyclodextrin and nystatin on anti-LacCer antibody-induced Lyn activation.

DIM prepared from neutrophil plasma membrane was diluted and incubated without or with 10 mM methyl-β-cyclodextrin (β-cyclodextrin) or 60 μg/ mL nystatin (nystatin) for 30 minutes at 25°C. After incubation, the reaction mixtures were ultracentrifuged and the pellets were resuspended in 5 mL of the kinase buffer, followed by incubation in normal IgM– or anti-LacCer IgM T5A7–coated dishes for 15 hours at 4°C. Then an aliquot of 50 μCi [γ³²P]-ATP was added to each dish, and the mixture was further incubated at 37°C for 5 minutes. The reaction was stopped by placing on ice. In some experiments, anti-LacCer–induced Lyn activation was assessed in the presence of 100 nM PP1 (PP1). (A) Phosphorylation of Lyn was analyzed by immunoprecipitation with mouse anti-Lyn IgM, followed by SDS-PAGE and blotting onto PVDF membrane and autoradiography (autoradiogram). To evaluate the recovery of Lyn, the blotted membrane was probed with anti-Lyn rabbit IgG (Immunoblot). (B) Lyn activity is expressed as fold increase compared with that of DIM incubated in normal IgM–coated dishes (normal IgM). Each bar shows the mean ± SD of independent 3 experiments.