Defective S1P1 expression in B cells from p66Shc^−/− mice. (A-B) qRT-PCR analysis of S1P1 (A) or S1P2 (B) mRNA in purified splenic B cells from wild-type (+/+) or p66Shc^−/− mice. The relative abundance of gene transcripts was determined on triplicate samples from at least 6 wild-type or p66Shc^−/− mice using the ddCt method and is expressed as the normalized fold expression (mean ± SD). (C) Flow cytometric analysis of surface S1P1 on splenic, LN, or PB B cells (analysis on gated CD22⁺ cells) from wild-type (+/+) or p66Shc^−/− mice. The data are expressed as the mean fluorescence intensity (MFI) ± SD (n ≥ 9 mice/group). Specificity controls for each experiment included a sample incubated with secondary Ab alone and a sample preincubated with FTY720 to induce receptor down-regulation. ***P < .001; **P < .01; and *P < .05 by Mann-Whitney rank-sum test. (D) Migration of purified splenic B cells from wild-type (+/+) or p66Shc^−/− mice, measured after treatment for 3 hours with S1P (100nM). The data, obtained on duplicate samples from at least 3 independent experiments, are presented as the mean migration index ± SD (ie, the ratio of migrated cells in S1P-treated samples vs untreated samples). ***P < .001.