p66Shc controls S1P1 expression in B cells. (A,C) qRT-PCR analysis of S1P1 (A) or S1P2 (C) mRNA in MEC B cells stably transfected with either empty vector (MEC ctr) or an expression construct encoding p66Shc (MEC p66). The relative abundance of gene transcripts was determined on triplicate samples from at least 3 independent experiments using the ddCt method and is expressed as the normalized fold expression (mean ± SD). (B,D) Flow cytometric analysis of surface S1P1 (B) or S1P2 (D) on MEC B cells stably transfected with either empty vector (MEC ctr) or an expression construct encoding p66Shc (MEC p66). The data in the histograms are expressed as the mean fluorescence intensity (MFI) ± SD. Representative FACS profiles of S1P1 (B) or S1P2 (D) are shown. Specificity controls for each experiment included a sample incubated with secondary Ab alone (α-mouse) and, for S1P1 stainings, a sample preincubated with FTY720 to induce receptor down-regulation. (E) Migration of the control and p66Shc-expressing MEC transfectants 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; and **P < .01.