FIG. 6.

Normal TRAF-, Vav1-, and OBF-1-mediated responses in Siah2 mutant mice and primary cells. (A) B-cell proliferation. Splenic white blood cells from 8-week-old wild-type or Siah2^−/− mice were activated in vitro for 72 h with LPS (0.5, 1, 2, and 20 μg/ml) or anti-IgM (5 and 20 μg/ml) or by culture on irradiated 3T3 fibroblasts expressing CD40L (1,000 and 2,000 irradiated cells per well). Proliferation was analyzed by [³H]thymidine incorporation for the final 16 h of culture. Data represent the means + standard deviations (SD) (error bars) of triplicate activations from each of three mice of each genotype. (B) T-cell proliferation was analyzed as in panel A after stimulation of splenic white blood cells in 96-well plates with ConA (0.5 μg/ml) or by culture in wells coated with anti-CD3 (5 and 10 μg/ml) or anti-CD3 (5 μg/ml) plus anti-CD28 (10 μg/ml) antibodies. (C and D) Wild-type (•) or Siah2^−/− (○) mice received intraperitoneal injections with 10 μg of the T-independent antigen NP-LPS (C) or with 100 μg of the T-dependent antigen NP-KLH precipitated on alum (D). Serum samples were obtained at time points after immunization and analyzed using an anti-NP and IgM-specific ELISA (C) or an anti-NP and IgG1-specific ELISA. Each circle in panel C represents the absorbance reading obtained from a 1:1,585 dilution of serum from an immunized mouse (the absorbance reading was linear with respect to dilution over the range 1:500 to 1:5,000), and each circle in panel D represents the absorbance reading obtained from a 1:10,000 dilution of serum from an immunized mouse (the absorbance reading was linear with respect to dilution over the range 1:3,170 to 1:31,700). Mean values are shown by horizontal lines. (E) Confluent BMM pooled from two wild-type or Siah2^−/− mice were starved of M-CSF for 24 h prior to stimulation for a further 24 h with recombinant CSF-1 (10,000 U/ml) alone or in combination with LPS (0.1 μg/ml), poly(I) · poly(C) (pI.pC, 0.1 or 50 μg/ml), TNF-α (1 or 100 ng/ml), or IFN-β (100 U/ml). DNA synthesis was measured by incorporation of [³H]thymidine for the final 8 h of culture. Data represent means + SD (error bars) of triplicate stimulations.