JE mRNA and protein are expressed in both mature and immature B cells and inhibit migration of immature B cells by interacting with CCR2. (A) Mature (IgD⁺ B220⁺) and immature (IgD^– B220⁺) cells from control mice were purified. Total RNA was isolated, and reverse transcription was carried out by using Superscript II RT. (B) Western blot analysis. Levels of JE in total cell lysates of IgD⁺ 220⁺ and IgD^– B220⁺ control cells. The band representing JE is indicated. The results presented are representative of 3 separate experiments. (C) Transwell migration. IgD^– B220⁺ cells were isolated from control or CCR2^–/– mice and pretreated for 30 minutes in 10 mL medium with or without JE. The cells were then washed and placed in the upper well of a 24-well transwell plate in the presence of SDF-1. The number of migrating cells found in the lower chamber was evaluated after 3 hours by FACS analysis. Migration was calculated as described in Figure 2, and the inhibition of migration was calculated as the migration with JE/migration without JE × 100. The results presented are representative of 3 different experiments. Error bars represent the standard error of the results of all the experiments used to calculate the average. *Significant at a 98% level.