Fig. 6.

The Dcp1 protein forms a complex with Ago2 and RBM4 in primary macrophages from septic mice and is associated with TNFα and IL-6 but not IL-10 mRNA. Sepsis was induced by CLP. Peritoneal macrophages were harvested from sham and septic mice that were moribund and sacrificed at days 10–28 (i.e. during late sepsis response). Cells were stimulated with 100 ng/ml of LPS for the indicated times. a Whole cell lysates were prepared and immunoprecipitated with anti-Dcp1 or IgG isotype antibody, resolved by SDS-10% PAGE, and then immunoblotted with a Dcp1, Ago2 or RBM4 antibody. The quantitation of protein levels is shown on the right. Values are presented as the fold change relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as the mean ± SD from three experiments. * p < 0.05, compared with 0 h. b RNA was extracted from the immunoprecipitated protein complexes and then analyzed by RT-PCR for the presence of TNFα, IL-6 or IL-10 RNA. RNA was also extracted from a portion of the cell lysate before the immunoprecipitation and was used to measure the level of the 18S rRNA as an internal (input) control. Cells were pooled from 8 sham mice and 6 late septic mice. The quantitation of RNA levels is shown on the right. Values were normalized to the 18S rRNA and are presented as the fold change relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as the mean ± SD from three experiments. * p < 0.05.