ZAP-70 activation delays IgM and CD79b internalization. (A) Stimulation of Ramos transfectants was performed with 5 μg/mL F(ab′)2 anti-IgM for 10 and 60 minutes at 37°C and stopped at 4°C. Density of IgM and CD79b on surface was analyzed by flow cytometry at the indicated time points with IgM-PE and CD79b-PE antibodies (top panel). The internalization kinetics of IgM differed from that of CD79b. The expression of ZAP-70 delayed the internalization of both IgM and CD79b (bottom panel). (B) Confocal microscopy (original magnification ×63). Ramos transfectants were stimulated with 5 μg/mL F(ab′)2 anti-IgM, and sequential images at 0, 10, 30, and 60 minutes after IgM-PE labeling were obtained. Density of surface IgM in Ramos GFP-ZAP-70 cells was higher than in Ramos GFP cells. Scale bar represents 5 μm.
