p66Shc controls S1P1 expression through its pro-oxidant activity. (A) Immunoblot analysis of Shc expression in MEC B cells stably transfected with empty vector (MEC ctr) or an expression construct encoding either wild-type p66Shc (MEC p66) or the S36A (MEC p66SA) or EE132/133QQ (MEC p66QQ) mutants. A control anti-actin blot of the stripped filter is shown below. The migration of molecular mass markers is indicated. The domain structure of p66Shc highlighting the location of the amino acid residues substituted in the mutants is schematized at the top of the panel. (B) Flow cytometric analysis of ROS production in the MEC B-cell transfectants loaded with the ROS-sensitive probe CM-H₂DCFDA. The data are expressed as relative ROS production using as a reference control (empty vector) MEC cells (n = 4). (C) Flow cytometric analysis of surface S1P1 in the MEC B-cell transfectants. The data are expressed as the mean fluorescence intensity (MFI) ± SD (n = 4). Specificity controls for each S1P1 staining experiment included a sample incubated with secondary Ab alone and a sample preincubated with FTY720 to induce receptor down-regulation. (D-E) Flow cytometric analysis of S1P1 mRNA in control MEC cells treated for 24 hours with 400μM H₂O₂ (D) and in p66Shc-expressing MEC cells treated for 24 hours with 0.2mM Trolox (E). Under these conditions, a 3-hour treatment with H₂O₂ increased intracellular ROS in control (empty vector) MEC by 2-fold, whereas Trolox reduced intracellular ROS in p66Shc-expressing MEC cells by 65%. ***P < .001; **P < .01; and *P < .05.