Figure 7.

AEBSF, a site 1 protease inhibitor, blocks tunicamycin and 2-deoxyglucose induced IRF3 phosphorylation and tunicamycin-dependent synergistic IFN-β induction. A) RAW cells were pre-treated with 300 μM AEBSF for 1h, then stimulated with 1μM Tg for 2h, 20mM 2DG for 5h, or 10 μg/mL Tm for 5h. Cells were fixed and then stained with anti-pIRF3 (S386) plus secondary anti-rabbit Alexa Fluor488. Results are representative of 2 independent experiments. B) RAW cells were pretreated with AEBSF as in (A) and then untreated (NT), stimulated with Tg 1h, or Tm 5h, followed by an additional 3h media or LPS as indicated. Whole cell lysates were resolved by SDS PAGE and immunoblotted with anti-pIRF3 (S396), IRF-3 or actin. Results are representative of 2 independent experiments. C) RAW cells were pretreated with AEBSF as in (A), then stimulated with 1h Tg or 5h Tm followed by an additional 3h LPS as indicated. Relative IFN-β mRNA was quantified by qPCR with normalization to 18S rRNA. Results were combined from 4-5 independent experiments and error bars represent the standard error of the mean. * P<0.007. D) Cells were stimulated as in (B). Whole cell lysates were resolved by SDS PAGE and immunoblotted for precursor (P) and mature (M) cleaved forms of ATF6. Results are representative of 3 independent experiments.