ZAP-70 enhances IgM-BCR signaling but does not participate in IgD-BCR signaling. (A) Ramos stable transfectants were stimulated with 5 μg/mL F(ab′)2 anti-IgM for 5 minutes and 24 hours. Enhanced Syk, Akt, and ERK1/2 phosphorylation was observed in IgM-BCR stimulated ZAP-70–expressing cells. (B) Immunoblotting analysis of Raji transfectants expressing constitutive phosphorylated ZAP-70 and ERK1/2. Ramos and Jurkat cells treated with pervanadate (PV) were used as positive controls. (C) Ramos stable transfectants were stimulated with 5 μg/mL F(ab′)2 anti-IgD for 5 minutes. On IgD stimulation, activation of Akt, but not of ERK1/2 or ZAP-70, was observed. Jurkat cells treated with PV were used as positive control. (D) Confocal microscopy (original magnification ×63). Ramos GFP-ZAP-70 cells were activated with 20 μg/mL F(ab′)2 anti-IgM or IgD for 40 minutes and then stained with anti–IgM-PE or anti–IgD-PE. ZAP-70 was translocated from the cytoplasm to the membrane and remained on surface after IgM activation while IgM internalization was almost complete (top panel). In contrast, while IgD was also internalized, no mobilization of ZAP-70 was observed after IgD stimulation (bottom panel). The scale bar in the image represents 5 μm.
