RBL-2H3 cells were sensitized with anti-DNP IgE, pretreated with 100 μg/ml piceatannol for 2 h at 37°C and stimulated or not (-) with Ag (1 μg/ml) for 5 min in the presence of the inhibitor. Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated Abs. (B and C) Syk kinase activity is required for Hrs tyrosine phosphorylation and ubiquitination. Clones (2 × 107) obtained by stable transfection of a Syk-negative variant of the RBL-2H3 cells with wild type Syk (Syk+) or a kinase-inactive form of Syk (KI) were sensitized AP24534 chemical structure with anti-DNP IgE and stimulated or not (-) with Ag (1ìg/ml) for 5 min. Cell lysates were immunoprecipitated with anti-Hrs
polyclonal Ab, resolved by SDS-PAGE and immunoblotted with the indicated Abs. The intensity of phosphorylated Hrs, normalized to Hrs level, was referred to the respective unstimulated samples. Mr are given in kilodaltons. Results shown are representative of three independent experiments. Supplementary Figure 5. Inducible
Hrs phosphorylation and ubiquitination does not affect protein stability. (A) RBL-2H3 cells were sensitized with anti-DNP IgE, pretreated with 25 μM cycloheximide for 2 h at 37°C and stimulated or not (-) with Ag (1 μg/ml) in the presence of the inhibitor for the indicated lengths of times. Total cell lysates were subjected to SDSPAGE and immunoblotted with the indicated Abs. The relative Syk protein amount, normalized with the band intensity of actin, was referred PD0332991 molecular weight to the unstimulated samples. Mr are given in kilodaltons. (B) Bar graph depicts estimations of Hrs protein amount after normalization with actin, expressed in relative units, 1 being the value given to the unstimulated samples (mean ± SD, n = 3). Differences were not significant (p > 0.05). “
“Tumor growth coincides with an accumulation of myeloid-derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b+CD115+Ly6G−Ly6Chigh MDSCs and granulocytic CD11b+CD115−Ly6G+Ly6Cint polymorphonuclear
(PMN)-MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8+ T-cell activation — including cytokine production, surface marker expression, survival, and cytotoxicity — is currently unclear. Tryptophan synthase Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen-driven CD8+ T-cell proliferation, but differ in their dependency on IFN-γ, STAT-1, IRF-1, and NO to do so. Moreover, MO-MDSC and PMN-MDSCs diminish IL-2 levels, but only MO-MDSCs affect IL-2Rα (CD25) expression and STAT-5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN-γ production by CD8+ T cells on a per cell basis, illustrating that some T-cell activation characteristics are actually stimulated by MDSCs. Conversely, MO-MDSCs counteract the activation-induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation.