Fig 2: IFP35 serves as a DAMP and stimulates the inflammatory responses. a, b Immunoblotting a and ELISA analysis b of IFP35 in the supernatant and cytoplasmic fraction of mouse RAW264.7 cells (ATCC TIB-71™), treated with LPS (1 µg ml-1). c, d Mice (n = 5 for each group) were intraperitoneally injected with LPS from E. coli (100 ng ml-1, c) or live S. typhimurium (1 × 104 CFU per mouse, d). The serum concentrations of IFP35 in mice were determined by ELISA. Data are presented as the mean ± s.e.m. Significance was tested by unpaired Student’s t-test (**P < 0.01). e TNF level in the supernatants of human THP1 cells (ATCC TIB-202™) was analyzed by ELISA 8 h post incubation with LPS (100 ng ml-1) or different purified proteins (5 µg ml-1 respectively). Data are presented as the mean ± s.e.m of three independent experiments. **P < 0.01. f The luciferase activity of HEK293T cells (ATCC CRL-11268™), transiently transfected with the genes of hTLR4–MD2–CD14 and NF-?B promoter with luciferase, and stimulated with 5 µg ml-1 hIFP35?N for 4 h (in the presence or absence of 25 µg ml-1 polymyxin B (PMB)). 100 ng ml-1 LPS was administrated as positive control. Data are presented as the mean ± s.e.m. of three independent transfections and are given as the ratio of luciferase activity relative to unstimulated cells. Significance in d and e was tested by one-way ANOVA followed by Student–Newman–Keuls test. **P < 0.01. g The survival rate of wild type (n = 14) and Ifp35 -/- mice (n = 14) intraperitoneally injected with LPS (50 µg kg-1) and d-gal (1 g kg-1). Significance was tested by log-rank test.*P < 0.05

Fig 3: Design and in vitro testing of FOXP2 transgenic constructs. (A) Schematic of the expression construct used to overexpress FoxP2. We expressed the P. discolor FoxP2 under the control of the human ubiquitin promoter (hUBC). To facilitate detection, we fused FoxP2 with a series of small peptidic tags (HIS‐tag, FLAG‐tag, T7, and Xpress®), and we used a T2A system to separately express the FoxP2 protein from the same promoter driving GFP expression. The GFP protein acted as a marker of the region of infection in the brain. To stabilize the transcript and enhance expression, we used the termination and polyadenylation signal of the human growth hormone (hGH). A premade virus coding for GFP under the control of a CMV promoter was purchased from Virovek (Hayward, California) (AAV5‐CMV‐GFP) to be used as a control. (B) In‐vitro testing of the expression cassette. We transfected HEK293T/17 cells (ATCC, CRL‐11268) with the FoxP2 expression cassette from panel (A) and detected strong overexpression of FoxP2 via western blot compared to untransfected HEK293T/17 cells. The full membrane image is shown in Figure S2. (C) Subcellular localization of the ectopically expressed FoxP2. We transfected HEK293T/17 cells (left panel, DAPI stain) with the FoxP2 expression cassette from panel (A) and detected strong overexpression of FoxP2 (middle panel) in IF using a FoxP2 antibody. GFP (right panel) indicates the presence of transgenic rather than endogenous protein in these cells. FoxP2 expression in the nucleus shows the expected localization of the transgenic protein.