RA modulates the activity of early signaling pathways in CpG-stimulated CD27⁺ B cells. (A) CD27⁺ B cells were treated with CpG (1 μg/mL) in the presence or absence of RA (100 nM). At indicated time points, total-cell extracts were prepared, and protein levels were determined by Western blot analysis. Lane 1 indicates medium control, and for lane 12, cells were treated with RA alone. (B) CD27⁺ B cells (0.5 × 10⁶/mL) were pretreated with U0126 (1, 3, 10, or 20 μM), SB202190 (0.5, 1, 2.5, or 5 μM), or Bay11-7082 (0.5, 1, 2, or 4 μM) for 60 minutes prior to stimulation with CpG (1 μg/mL) in the presence or absence of RA (100 nM), and thymidine incorporation was determined. The data represent mean cpm ± SEM of 5 independent experiments. (C) CD27⁺ B cells (1 × 10⁶/mL) were pretreated with U0126 (U, 10 μM), SB202190 (S, 0.5 μM), or Bay11-7082 (B, 2 μM) for 60 minutes prior to stimulation with CpG (1 μg/mL) for 24 hours in the presence or absence of RA (100 nM). Total-cell extracts were subjected to Western blot analysis and examined for cyclin D3 expression. One representative experiment is shown. (D) CD27⁺ B cells (0.5 × 10⁶/mL) were pretreated with SB202190 (0.5 μM) and cultured as described in panel B. Cell supernatants were collected at day 3 and subjected to ELISA specific for IL-10. The results represent mean IL-10 secretion ± SEM of 4 independent experiments. (E) CD27⁺ B cells were treated as in panel D, and supernatants were harvested at day 5 and subjected to ELISA specific for IgG. The data represent mean IgG secretion ± SD of 2 independent experiments. S indicates SB202190.