Journal: iScience

Article Title: Alphaherpesvirus manipulates retinoic acid metabolism for optimal replication

doi: 10.1016/j.isci.2024.110144

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-p-IRF3 , Cell Signaling Technology , Cat# 29047 RRID: AB_2773013.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: Acta pharmacologica Sinica

Article Title: Probiotics protect against RSV infection by modulating the microbiota-alveolar-macrophage axis.

doi: 10.1038/s41401-020-00573-5

Figure Lengend Snippet: Fig. 6 Probiotic pretreatment promoted IFN-β production in AMs. a Isolated AMs were pretreated with acetate and then stimulated with RSV (1 MOI) at the indicated time points. The levels of IFN-β were tested by qPCR. The results are from three independent experiments. b Quantification of IFN-β in sorted AMs from all groups of mice by qPCR (n=4). c Quantification of IFN-β in lungs from all groups of mice by qPCR (n=4). d Levels of IFNβ in serum from all groups of mice were examined by ELISA (n=4). e IFNAR1 was blocked using a neutralizing antibody in RSV-infected mice, and we then evaluated the effects of the probiotics on RSV infection. RSV mRNA expression in the lungs was examined using qPCR at 3 days after RSV infection (n=4). ɑIFNAR, antibody against IFNAR. f For the depletion of macrophages, the mice were intranasally inoculated with a single dose of 100 µL of clodronate, and we then evaluated the effects of the probiotics on RSV infection. RSV mRNA expression in lungs was examined using qPCR at 3 days after RSV infection (n=4). g FACS analysis of phosphorylated TBK1 (Ser172) in AMs at 24 h after RSV infection. h FACS analysis of phosphorylated IRF3 (Ser396) in AMs at 24 h after RSV infection. All results are expressed as the mean±SD (a–f). *P<0.05, **P<0.01, ***P<0.001 by Student’s t test.

Article Snippet: Rabbit polyclonal antibodies against phospho-IRF3-PE (Ser396, #29047), phosphoTBK1 (Ser172, #5483), and IFNβ (#97450 s) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Infection, Probiotics, Expressing

Journal: Virology

Article Title: MicroRNA-23 inhibits PRRSV replication by directly targeting PRRSV RNA and possibly by upregulating type I interferons.

doi: 10.1016/j.virol.2013.12.020

Figure Lengend Snippet: Fig. 6. miR-23 increases type I interferon expression through IRF3/IRF7 activation during PRRSV infection. (A, B and C) IRF3/IRF7 inhibitor impaired the induction of type I IFNs during PRRSV infection. Transfection of miR-23 mimics or NC in PAMs was performed prior to the treatment of the indicated inhibitors or DMSO, followed by PRRSV JXwn06 infection for 48 h (MOI¼0.01). Cells were then harvested for quantifying the expression of type I interferon genes IFN-β (A) and IFN-α (B), and ORF7 (C) using qRT- PCR, normalized to GAPDH. The data were represented as fold changes of the indicated genes after over-expression of miR-23 (NC was set up as 1 and not shown in the figure). Statistical significance was analyzed by one-way ANOVA followed by post hoc Dunnett0s multiple comparison. Significance compared to DMSO-baseline: nPo0.05. (D, E, F, and G) miR-23 increases activation of IRF3 during PRRSV infection. Either miR-23 or NC mimics and pRL-TK were co-transfected with IRF3 (D), ISRE (E), or NF-κB (F) luciferase reporters into Marc-145 cells. Cells were then either infected with CH-1a (MOI¼0.01) 6 h after transfection, or transfected with 1 μg poly(I:C) 24 h post trasfection, or left untreated (mock). All cells were harvested 36 h post-transfection for dual-luciferase assay. (G) Western blot analysis of phospho-IRF3 or IRF3 in PAMs transfected with miR-23 mimics followed by infection with JXwn06 (MOI¼0.01) for 48 h. β-actin was shown as a loading control. (H) miR-23 plays no role in IFN down- stream pathway. Luciferase activity in lysates of CRL-2843 cells co-transfected with ISRE luciferase reporter, pRL-TK and miRNA mimics for 6 h and incubated with or without 10 U/ml IFN-α for 24 h. The data were represented as fold increase of ISRE. Data were representative of three independent experiments (mean7SD). Statistical significance was analyzed by t-test; nPo0.05; ns, not significant.

Article Snippet: Membranes were blocked with 5% milk in PBS with Tween-20 (PBST 0.025% Tween-20) and probed with Rabbit anti-GP5 polyclonal antibodies (1:5000, prepared in our lab), anti-Phospho-IRF3 polyclonal antibodies (1:1000; #4947, Cell Signaling), or anti-IRF3 polyclonal antibodies (1:1000; #119041, Cell Signaling) for 1 h at room temperature.

Techniques: Expressing, Activation Assay, Infection, Transfection, Quantitative RT-PCR, Over Expression, Comparison, Luciferase, Western Blot, Control, Activity Assay, Incubation