STAT1 is required for full expression of Ttp in LPS-treated primary macrophages. (A) Macrophages derived from bone marrow (BMM) of STAT1WT and STAT1KO mice were left untreated or treated with IFN-γ (g), LPS (L), or both (L/g) and Ttp mRNA induction was measured by qRT-PCR and normalized to samples from untreated cells. Error bars indicate SD, n = 3 experiments. (B) BMMs isolated from STATWT mice were treated as explained for panel A, except the time of the treatment was 3 hours instead of 1 hour. TTP protein levels were analyzed by Western blotting of whole-cell extracts using a TTP antibody. The blot was reprobed with a panERK antibody to control for equal protein loading. (C) BMMs from STAT1WT and STAT1KO mice were treated for 1 hour and 4 hours with LPS or left untreated, and mRNA induction was analyzed as described in panel A. Error bars indicate SD, n = 3 experiments. (D) Same cells as those used in panel C were treated for 2 and 4 hours with LPS or left untreated. TTP protein was detected by Western blotting of whole-cell extracts using a TTP antibody. Activation of IFN signaling by endogenous production of type I interferon in LPS-treated macrophages was demonstrated using antibody to tyrosine-phosphorylated STAT1 (pY701-S1). Equal protein loading was confirmed using a STAT1 antibody and a panERK antibody. (E) BMMs from IFN-β knockout (IFNbKO) and wild-type controls (IFNbWT) were stimulated with LPS for the times indicated. Total RNA was isolated and qRT PCR was performed. Error bars indicate SD, n = 3 experiments. (F) Whole-cell extracts of the same cells as used in panel E were stimulated with LPS as indicated. TTP protein was detected by Western blotting using a TTP antibody.