GEP analysis of IRF4-deficient and wild-type B cells. (A) RNA was isolated from splenic irf4^−/− and irf4^+/+ B cells purified by magnetic cell separation and processed for hybridization on microarrays arrays (B cell fractions were isolated separately from four mice per genotype and independently processed for microarray hybridization). Gene expression differences between irf4^+/+ and irf4^−/− B cells were determined by supervised analysis (n = 4 each group). Color changes within a row indicate expression levels relative to the mean of the sample population. Values are quantified by the scale bar that visualizes the difference in the z_ge score (expression difference/SD) relative to the mean (0). Genes are ranked according to their z_g score (mean expression difference of the respective gene between IRF4-deficient and wild-type samples/SD). The gene expression differences among the genotypes are shown in brackets and grouped according to functional or operational categories: [a] cytoskeleton/membrane function, [b] unknown, [c] cellular trafficking, [d] protein degradation, [e] transporter protein, [f] cell membrane protein, [g] metabolism, and [h] translation. Genes that showed the same trend of expression compared with a published GEP analysis of MZ versus FO B cells are underlined. (B) Total RNA from irf4^+/+ and irf4^−/− splenic B cells was analyzed for expression of RGS13, ALCAM, CXCR7, and PLXND1 by qRT-PCR. The expression was corrected for β-actin levels and then normalized to wild-type B cells. Data are shown as mean ± SD (n = 3, two independent experiments). Statistical significance was analyzed by Student’s t test (**, P < 0.01; ***, P < 0.001). (C) ALCAM and CXCR7 expression on IgM⁺ splenic B cells from irf4^+/+ and irf4^−/− mice was assessed by flow cytometry. Data are shown as mean ± SD (n = 6, two independent experiments). Statistical significance was determined by Student’s t test (**, P < 0.01).