Figure 4. Nuclear localization of STRAP is linked to NF-κB activation during the late phase of LPS-stimulation.

(a) LPS induces the translocation of STRAP to the nucleus at 4 h. STRAP was examined by an immunofluorescence assay (IFA) using an anti-STRAP antibody, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 5 μm. (b) STRAP translocates to the nucleus in response to LPS at 4 h. Endogenous STRAP was analyzed in the cytosolic and nuclear fractions of LPS-stimulated RAW cells by immunoblotting. Tubulin and lamin A/C were used as loading controls for the cytosolic and nuclear fractions, respectively. (c) STRAP endogenously interacts with p65 in response to LPS at 4 h. RAW cells were treated with LPS (80 ng/ml) at different time points. Cell lysates were immunoprecipitated with anti-p65 antibody and immunoblotted with anti-STRAP or anti-p65 antibodies. (d) STRAP and p65 localizes to the nucleus at 4 h following LPS-stimulation. STRAP and Myc-p65 were visualized by IFA with anti-STRAP and anti-Myc antibodies, and the nuclei were stained with DAPI. Scale bars, 5 μm. (e) STRAP enhances the transcriptional activity of IL-6. Mouse embryonic fibroblasts (MEFs) were transfected with an empty vector (Mock) or GFP-STRAP vector (GFP-STRAP) along with the IL-6 luciferase reporter and Renilla reporter and incubated with LPS (80 ng/ml) for 4 h. *P < 0.01 and **P < 0.005 (Student’s t-test). Data are representative of three independent experiments and are presented as mean ± s.d. in (e).