Fig. 1:

PIP₂ is hydrolyzed in all B cell subsets following anti-IgM stimulation. (A) : Freshly isolated MACS-enriched wt B cells were fixed with 2% paraformaldehyde, permeabilized and labeled with antibodies directed against CD19, CD21, and HSA for B cell subset identification. (B): B cells from (A) were examined for basal PIP₂ levels utilizing an anti-PI(4,5)P₂ antibody. Data is an example of multiple experiments. (C): B cells were stimulated for the indicated times with 20 μg/ml anti-IgM, immediately fixed and permeabilized, then labeled and examined for PIP₂ levels as in (B); T1 (filled diamonds), T2 (filled squares), mature FoB (open circles), preMZ (filled triangles), mature MZB (open triangles). Data is displayed as the average MFI of duplicate samples, and is a representative of at least 4 experiments. (D): The specificity of the anti-PIP₂ antibody was tested by pre-incubation of the antibody with PIP₂ liposomes for 15 min prior to intracellular labeling of MACS-enriched total B cells as in (B).