Figure 6
Figure 6. Hyperresponsive phenotype of SHD1−/− T, B, and bone marrow cells. (A) Splenic T cells from wild-type, SHD1+/−, or SHD1−/− mice were stimulated with the indicated concentrations of anti-CD3 in the presence of anti-CD28 (1 μg/mL). Proliferation was assessed by the 3H-thymidine uptake after 72 hours of stimulation. Each assay was conducted in triplicate and the data are representative of 4 experiments. The data are the mean plus or minus SD. (B) The IL-2 production by splenic T cells from wild-type, SHD1+/−, or SHD1−/− mice. The culture supernatants from panel A were analyzed by ELISA for IL-2 concentration. The data are the mean plus or minus SD (n = 3). (C) Cultured splenic T cells from wild-type or SHD1−/− mice were starved, and then stimulated with IL-2 (10 ng/mL) for 10 or 30 minutes. The cells were lysed and subjected to immunoprecipitation and a Western blot analysis for the phosphorylation of STAT5. (D) IgM production by B cells. Purified splenic B cells from wild-type, SHD1+/−, or SHD1−/− mice were stimulated with the indicated concentrations of IL-5 plus anti-CD40 (1 μg/mL) for 7 days. The concentration of IgM in the culture supernatants was analyzed by ELISA. The data are the mean plus or minus SD (n = 3). (E) BM cell proliferation. BM cells from wild-type, SHD1+/−, or SHD1−/− mice were cultured in the media containing IL-3 and GM-CSF. Cell numbers were enumerated every day as described in “Proliferation assays for bone marrow cells and mast cells.” The data are the mean plus or minus SD (n = 3). (F) Proliferation of mast cells. Mast cells derived from SHD1-mutant mice were cultured in the presence of 1 or 10 ng/mL IL-3, and the cell numbers were enumerated at the indicated time points (mean ± SD, n = 3). (G) Induction of STAT5 target genes in wild-type, SHD1+/−, or SHD1−/− mice. Total RNA was extracted from cultured T cells stimulated with the indicated concentrations of IL-2 for the indicated times. Quantitative RT-PCR analysis was performed as described in “RT-PCR.” Data are shown as a ratio compared with the average value of prestimulated (0 minutes) wild-type in each graph.

Hyperresponsive phenotype of SHD1−/− T, B, and bone marrow cells. (A) Splenic T cells from wild-type, SHD1+/−, or SHD1−/− mice were stimulated with the indicated concentrations of anti-CD3 in the presence of anti-CD28 (1 μg/mL). Proliferation was assessed by the 3H-thymidine uptake after 72 hours of stimulation. Each assay was conducted in triplicate and the data are representative of 4 experiments. The data are the mean plus or minus SD. (B) The IL-2 production by splenic T cells from wild-type, SHD1+/−, or SHD1−/− mice. The culture supernatants from panel A were analyzed by ELISA for IL-2 concentration. The data are the mean plus or minus SD (n = 3). (C) Cultured splenic T cells from wild-type or SHD1−/− mice were starved, and then stimulated with IL-2 (10 ng/mL) for 10 or 30 minutes. The cells were lysed and subjected to immunoprecipitation and a Western blot analysis for the phosphorylation of STAT5. (D) IgM production by B cells. Purified splenic B cells from wild-type, SHD1+/−, or SHD1−/− mice were stimulated with the indicated concentrations of IL-5 plus anti-CD40 (1 μg/mL) for 7 days. The concentration of IgM in the culture supernatants was analyzed by ELISA. The data are the mean plus or minus SD (n = 3). (E) BM cell proliferation. BM cells from wild-type, SHD1+/−, or SHD1−/− mice were cultured in the media containing IL-3 and GM-CSF. Cell numbers were enumerated every day as described in “Proliferation assays for bone marrow cells and mast cells.” The data are the mean plus or minus SD (n = 3). (F) Proliferation of mast cells. Mast cells derived from SHD1-mutant mice were cultured in the presence of 1 or 10 ng/mL IL-3, and the cell numbers were enumerated at the indicated time points (mean ± SD, n = 3). (G) Induction of STAT5 target genes in wild-type, SHD1+/−, or SHD1−/− mice. Total RNA was extracted from cultured T cells stimulated with the indicated concentrations of IL-2 for the indicated times. Quantitative RT-PCR analysis was performed as described in “RT-PCR.” Data are shown as a ratio compared with the average value of prestimulated (0 minutes) wild-type in each graph.

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