r848  (InvivoGen)

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: (A) Schematic of IRF7 activation, nuclear localization, and DNA binding downstream of multiple pattern recognition receptor pathways. (B) Overview of experimental approach employed in HEK Blue hTLR7 cells. C) Representative Western blot image of HEK Blue hTLR7 nuclear lysates following stimulation with R848 for 3-hours. β-actin and total H3 are included as loading controls. (D) Densitometry analysis of band intensities for risk and protective haplotype bands based on the quantitative Western blot presented in Figure S2. IRF7 bands were normalized to a total protein stain to calculate normalized signal as described in Methods. Welch’s t-test was used to determine significance. (E) Normalized ISRE luciferase activity (ratio of ISRE firefly to Renilla luciferase relative light units (RLU)) following 24-hours of R848 stimulation. Welch’s t-test was used to determine significance. (F) IFN-α measured in cell-free supernatants from cells stimulated with R848 for 3– and 24-hours. Data are mean ± SEM of triplicate samples (unless otherwise indicated) and are representative of at least three independent experiments. Significance was assessed with two-way ANOVA with a Holm-Šidak’s multiple comparisons correction. *P<0.05; ***P<0.005; ns indicates non-significant (P>0.05). ISRE: Interferon stimulated response element; EV: Empty Vector; R848: Resiquimod.

Article Snippet: Ligands used in cell stimulations include Resiquimod (R848) (Invivogen: 1ug/mL), polydA:dT-Lyovec (Invivogen: 2.5ug/mL), and Poly(I:C) LMW (Invivogen: 2.5ug/mL).

Techniques: Activation Assay, Binding Assay, Western Blot, Staining, Luciferase, Activity Assay, Plasmid Preparation

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: Using CRISPR targeting and homologous recombination, we mutated C/G to T/A at position 141,263,661 on chromosome 7 (Genome Reference Consortium Mouse Build 38), resulting in an R334Q substitution in mouse IRF7 that is analogous to the rs1131665 SLE risk-variant R412Q substitution in human IRF7. (A) 100 micrograms of resiquimod (R848) was epicutaneously applied three times per week to the ears of genome edited C57BL/6 mice with the lupus risk or non-risk IRF7 genotype for a total of eight weeks. (B) Sera was collected and used to quantify anti-double-stranded DNA (ds-DNA) antibodies by ELISA. (C) Eightweek-old male mice with the lupus risk or non-risk IRF7 genotype were intranasally infected with 1 million plaque-forming units of VSV-NJ. (D) After 24 hours, virus was quantified in lung homogenates using viral plaque assays. (E) Model for the lupus risk genotype in IRF7 enhancing both viral responses (with the lupus risk genotype enhancing viral clearance) and autoimmunity (with the lupus risk genotype enhancing the production of autoantibodies).

Article Snippet: Ligands used in cell stimulations include Resiquimod (R848) (Invivogen: 1ug/mL), polydA:dT-Lyovec (Invivogen: 2.5ug/mL), and Poly(I:C) LMW (Invivogen: 2.5ug/mL).

Techniques: CRISPR, Homologous Recombination, Variant Assay, Enzyme-linked Immunosorbent Assay, Infection, Virus