p66Shc modulates the IL-9–dependent expression of homing chemokines by stromal cells through its pro-oxidant activity. (A) Immunoblot analysis with anti-Shc antibodies of postnuclear supernatants from MEC1 B cells stably transfected with empty vector (MEC ctr) or an expression construct encoding WT p66Shc (MEC p66) or the EE132/133QQ (MEC QQ) mutant (n = 3). A control anti-actin blot of the stripped filter is shown below. The migration of molecular mass markers is indicated. (B) qRT-PCR analysis of IL-9 mRNA in MEC transfectants. The relative gene transcript abundance was determined from triplicate samples using the dCt method and normalized to HPRT1. (C) Quantification by ELISA of IL-9 released in the culture supernatants of MEC ctr (n = 5), MEC p66 (n = 5), or MEC QQ (n = 5) transfectants. (D) qRT-PCR analysis of CXCL9, CXCL10, CXCL11, and CCL2 mRNA in HS-5 human stromal cells cultured for 48 hours with conditioned supernatants (SN) from MEC transfectants (n = 4). (E) qRT-PCR analysis of CXCL9, CXCL10, CXCL11, and CCL2 mRNA in HS-5 human stromal cells cultured for 48 hours with conditioned supernatants (SN) from MEC transfectants, in the presence of 0.1 ng/mL isotype-control antibody (ctr Ab) or 0.1 ng/mL neutralizing anti–IL-9 mAb (IL-9 Ab). The relative gene transcript abundance was determined on triplicate samples using the dCt method and normalized to HPRT1. Data are mean ± standard deviation. ****P ≤ .0001, ***P ≤ .001, **P ≤ .01, *P ≤ .05, 1-way ANOVA with Tukey’s post hoc correction, multiple comparisons. ns, not significant.