Effect of IFI16 acetylation on IFN-β production during <i>de novo</i> HSV-1 infection in HFF cells The HFF cells uninfected or infected with HSV-1 (1 PFU/cell MOI) in the presence or absence of 1 μM C-646 for 30 min or 2 h were washed and incubated with or without 1 μM C-646 for 6 h.

<p><b>(A)</b> IFN-β in the culture supernatants was quantitated by ELISA. <b>(B and C)</b> Cells were examined by IFA with anti-pIRF-3 and Alexa Fluor-488 secondary antibodies. Insets in the merged panels are enlarged. The white and yellow arrows indicate the pIRF-3 in the cytoplasm or nucleus, respectively. Quantitation of nuclear pIRF-3 dots is shown in (C). <b>(D)</b> PLA with anti-IFI16 and anti-acetylated lysine antibodies. White and yellow arrows indicate the cytoplasmic and nuclear acetylated IFI16, respectively. <b>(E)</b> PLA with mouse and rabbit anti-IFI16 antibodies. White and yellow arrows indicate the cytoplasmic and nuclear IFI16, respectively. <b>(F)</b> WCL lysates in NETN buffer were IP-ed with anti-acetylated lysine and STING antibodies and immunoblotted for IFI16 and IRF-3. Proteins were also immunoblotted for total IFI16, pIRF-3, total IRF-3, and total STING with tubulin used as loading control. <b>(G)</b> HFF cells were transfected with control or STING Si-RNA with electroporation, either left uninfected or infected with HSV-1 (1 PFU/cell MOI) for 30 minutes or 2 h, washed and incubated for 6 h. WCL prepared in NETN buffer were western blotted for total STING, pIRF3, IRF3 and tubulin or IP-ed with anti-acetylated lysine antibodies and western blotted for IFI16 and tubulin, or IP-ed with anti-IFI16 antibodies and western blotted for IFI16. <b>(H)</b> HFF cells electroporated with control or STING Si-RNA were uninfected or infected with HSV-1 for 2 h, washed and incubated for 6h. Cell culture supernatants were used to measure the levels of IFN-β by ELISA.