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Figure 2. IFN-ß Induces SOCS-1 expression in macrophages, and ectopic expression of SOCS-1 inhibits recruitment of STAT-1
and RNA Pol II to the CD40 promoter, and modification of H3 and H4 histones in response to IFN-ß. A) RAW264.7 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 12 h, then total RNA was isolated and analyzed by RPA for SOCS/1 and GAPDH mRNA. B) RAW264.7 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 24 h. Protein lysates were subjected to immunoblotting with anti-SOCS-1 antibody, stripped and reprobed with anti-actin as a loading control. C) RAW264.7 cells were transiently transfected with 0.2 µg of the murine SOCS-1 promoter construct, treated with medium or IFN-ß (100 U/ml) for 8 h, and analyzed for luciferase activity. D) Protein lysates were prepared from RAW264.7 and RAW-SOCS-1 cells and subjected to immunoblotting with anti-SOCS-1 antibody for confirmation of ectopic SOCS-1 expression, stripped and reprobed with anti-actin as a loading control (E) RAW264.7 and RAW-SOCS-1 cells were treated with IFN-ß (100 U/ml) for up to 24 h, and CD40, IRF-1 and GAPDH mRNA was analyzed. F) RAW264.7 and RAW-SOCS-1 cells were incubated in the absence or presence of IFN-ß (100 U/ml) for up to 8 h, then the cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with antibodies against STAT-1, histone acetylation (Ac-H3, Ac-H4), RNA Pol II, phospho-Pol IISer5, or normal rabbit IgG.
