IL-3–induced RALDH2 expression in basophils is dependent on the activation of PI3-kinase and NF-κB signaling pathways. (A) Basophils were cultured for 24 hours in the presence or absence of different inhibitors: PI3-kinase inhibitor, LY2940002 (30 μM); NF-κB signaling pathway inhibitor, PDCA (50 μM); mTOR inhibitor, rapamycin (50 μM); and MEK inhibitor, U1026 (10 μM). Inhibitors were added 30 minutes prior to addition of IL-3 (10 ng/mL). Expression of RALDH2 and Pim1, as well as phosphorylation status of STAT5, S6, and ERK1/2, was analyzed by Western blotting using specific antibodies. Samples of freshly isolated basophils (f.i.) and basophils cultured without IL-3 for 24 hours (buffer) were included in the analysis. (B) Modulation of RALDH2 expression by the xenobiotics PMA and ionomycin. Basophils were cultured for 24 hours with or without IL-3 (10 ng/mL), retinal (5 nM), retinoic acid (RA, 10 nM), PMA (20 nM), and/or ionomycin (iono., 1 μM). RALDH2 expression was analyzed as described for panel A. Actin is shown as a loading control.