Figure 5

The C-terminus of STRAP is critical for IL-6 expression.

(a) Schematic model of STRAP and its C-terminal deletion mutant (GFP-STRAP ΔC) tagged with GFP. (b) GFP-STRAP ΔC is not sufficient to significantly enhance IL-6 production, in contrast to wild-type STRAP. RAW macrophages stably expressing Mock, GFP-STRAP, or GFP-STRAP ΔC were treated with LPS (80 ng/ml) or Pam3CSK4 (100 ng/ml) for 12 h. IL-6 levels were analyzed by ELISA. *P < 0.01. (c) The C-terminus of STRAP is involved in its subcellular localization. Macrophages stably expressing GFP-STRAP or GFP-STRAP ΔC were examined by IFA, and nuclei were stained with DAPI. Scale bars, 5 μm. (d) Nuclear translocation of GFP-STRAP ΔC in response to LPS. The presence of GFP-STRAP and GFP-STRAP ΔC in the nuclear fractions of LPS-stimulated RAW cells was analyzed by immunoblotting. Lamin A/C was used as loading controls for the nuclear fractions. (e) GFP-STRAPΔC was not as effective in promoting p65 phosphorylation as wild-type STRAP. The phosphorylation of p65 was analyzed in LPS-stimulated RAW cells stably expressing Mock, GFP-STRAP, or GFP-STRAP ΔC by immunoblotting. Expression levels of phosphorylated p65 were quantified by densitometry of bands and reported relative to tubulin. *P < 0.01 and **P < 0.005. (f) The C-terminus of STRAP is important for its association with TAK1. RAW cells stably expressing GFP-STRAP or GFP-STRAP ΔC were treated with LPS (80 ng/ml). Cell lysates were immunoprecipitated with an anti-TAK1 antibody and immunoblotted with anti-GFP or anti-TAK1 antibodies. Data are representative of three independent experiments and are presented as mean ± s.d. in (b,e).