M35 does not target signaling downstream of the type I IFN receptor.

<p>(A) 293T cells were co-transfected with a reporter plasmid containing the endogenous ISG56 promoter cloned upstream of the firefly luciferase gene, pRL-TK and V5-tagged versions of LacZ, M27 or M35. 24 hours post transfection, cells were stimulated by addition of 0.1 ng/ml recombinant human IFNβ or left unstimulated. At 16 hours post stimulation, cells were lysed for analysis of luciferase production (left panel). Simultaneously, 293T cells were co-transfected with expression constructs for cGAS (stimulated) or GFP (unstimulated) together with mCherry-STING, the IFNβ-Luc and pRL-TK luciferase plasmids, and V5-tagged versions of LacZ, M27 or M35. Cells were lysed at 20 hours post transfection for analysis of luciferase production (right panel). Luciferase fold induction was calculated based on luciferase values normalized to Renilla from stimulated samples divided by corresponding values from unstimulated samples. For both panels, data is combined from four independent experiments and shown as mean ± SD. (B) Immortalized BMDM stably expressing myc-tagged LacZ or M35 were stimulated by addition of 100 U/ml of recombinant murine IFNβ or left unstimulated. Cells were lysed for RNA extraction at indicated timepoints for analysis of ISG transcription by quantitative RT-PCR (left panel: CXCL10, right panel: IFIT3) and normalized to the housekeeping gene Rpl8. Data is shown as mean ± SD and combined from three independent experiments.