JE secreted by B cells inhibits immature B-cell actin polymerization and migration. (A) Actin polymerization. Immature B cells were isolated from Ii^–/– mice and pretreated for 30 minutes in 10 mL medium with or without JE. The cells were then stimulated with 50 μg/mL SDF-1 for 15 seconds, fixed, and permeabilized, and their intracellular F-actin was stained with FITC-phalloidin. The change in the polymerized actin was analyzed by FACS. Percentage increase in actin polymerization was calculated as described in Figure 2. (B) Transwell migration. Immature B cells were isolated from Ii^–/– mice and pretreated for 30 minutes with medium, JE (1 ng/mL), or SLC (1 μg/mL). The cells were washed and then assayed for migration as described earlier. The results presented are representatives of 5 different experiments. (C) FACS analysis of CXCR4 expression on Ii^–/– B cells incubated in the presence or absence of CCL2. Ii^–/– immature B cells were stained with anti-CXCR4 or a control antibody. The results presented are representative of 3 different experiments. (D) B220⁺ cells from control mice were isolated, and 2 × 10⁷ (concentrated) or 2 × 106 (diluted) cells were suspended in 10 mL medium for 30 minutes, and their conditioned medium was collected. Concentrated conditioned medium was treated overnight with or without anti-JE or control antibody. The treated and nontreated conditioned media were used for 30 minutes of pretreatment of immature B cells that were isolated from Ii^–/– mice. The pretreated immature B cells were then stimulated with 50 μg/mL SDF-1 for 15 seconds, fixed, permeabilized, and their intracellular F-actin was stained with FITC-phalloidin. The change in the polymerized actin was analyzed by FACS and calculated as described in Figure 2. Error bars represent the standard error of the results of all the experiments used to calculate the average. *Significant at a 98% level.