Figure 3.

Effects of NMDAR antagonists on B-cell signaling. A) Reduced Ca²⁺-flux in BCR-activated B cells in the presence of NMDAR antagonists. Indo-1 AM-labelled B cells were stimulated with α-IgM (10 μg/ml) in the presence or absence of ifenprodil (left) or memantine (right) and Ca²⁺-flux was determined by flow cytometry. Corresponding graphs show the mean + SD relative ΔCa²⁺-flux of three experiments. B-E) NMDAR antagonists attenuate BCR- and LPS-induced activation of important signaling molecules. B cells were activated with α-IgM (10 µg/ml) or LPS (10 µg/ml) or α-IgM plus CD40 Abs (5 µg/ml) in the presence or absence of ifenprodil (30 μM) in B) short-term and C-E) long-term stimulation. Activation of the indicated signaling proteins in B) total, C, E) cytoplasmic and D) nuclear protein extracts was analyzed by Western blot. β-Actin and Lamin B expression served as controls for protein loading. Indicated numbers give the relative protein expression after quantification and normalization to controls. Data are the representative of two (E) and three (B-D) independent experiments.