Hantaan Virus Infection Induces CXCL10 Expression through TLR3, RIG-I, and MDA-5 Pathways Correlated with the Disease Severity
Figure 5
p65 and IRF7 translocate to nucleus and bind to CXCL10’s promoter after HTNV infection. (a) WB results showed that HTNV infection induced the nuclear translocation of p65 and IRF7. Lysates were also immunoblotted using anti--tubulin or antihistone H3 (H3) as loading controls and to confirm the integrity of the cytosolic and nuclear lysates, respectively. (b) Semiquantitative analysis of p65 and IRF7 levels indicated in (a) was performed by Band-Scan software 5.0, and expression of -tubulin or H3 was used as cytosolic or nuclear control, respectively. Data were means ± SE. *, 24 h or 48 h versus 0 h p.i with HTNV. (c) At 24 h and 48 h p.i, phosphorylation of Ikk, IκB, and IRF7 can be detected by Western blot. (d) EMSA results showed direct bindings of p50 and p65 to the NF-κB1 site (left) and IRF7 to the ISRE site (right) of CXCL10 promoter after HTNV infection. Super shift bands were shown when the specific antibodies to p50, p65, and IRF7 were used, respectively. (e) ChIP assay also documented that p50, p65, and IRF7 bound directly to the CXCL10 promoter. The upper panel showed that, at 48 h post-HTNV infection, both a 177-bp DNA fragment and a 200-bp DNA fragment containing the NF-κB1 site and ISRE site in the CXCL10 promoter, respectively, can be amplified successfully, while the mock virus cannot (lower panel). The input DNA and the positive (anti-polymerase antibody) and the negative control (normal mouse IgG) were used to verify the reliability of ChIP assay.
