recombinant ifnβ  (R&D Systems)

Journal: bioRxiv

Article Title: Loss of Vpr-driven TRAIL-R2 expression protects HIV-infected cells from non-canonical NK cell TRAIL attack

doi: 10.64898/2026.04.15.718741

Figure Lengend Snippet: (A) Volcano plot of DEGs for the sorted infected populations with vs without NK cell exposure. Black dots indicate a p-adjusted value less than 0.05. Highlighted in orange are all the genes identified in the Hallmark “TNFA signaling via NF-κB” pathway. Highlighted in blue are all the genes identified in the Hallmark “Interferon gamma response” and “Interferon alpha response” pathways. See also Fig S6A for the expanded GSEA Hallmark analysis. (B and C) Detection of cytokines in co-culture. (C) NK cell expression of TNF-α and IFN-γ detected in mock vs infected cell co-cultures. See also Fig S6B and C for gating strategies. Summary data of TNF-𝘢 expression from 9 independent experiments (n=14 donors). Summary data of IFN-ɣ expression from 3 independent experiments (n=6 donors). (C) Detection of type I IFN via BST-2 surface expression on CD4 + T cells in cultures treated with recombinant IFN-β or in NK cell co-cultures. See also Fig S6D and E. Summary data from 3 independent experiments (n=6 donors). Statistical analysis for B and C: paired t test, **p<0.01, ***p<0.001, ****p<0.0001. (D) Cytokine-induced and NK cell co-culture changes in MHC-I on infected cells. Infected cultures were cultured overnight +/- 1µg/mL recombinant cytokines (TNF-𝘢, IFN-ɣ, or IFN-β) or +/- NK cells, followed by measurement of changes in HLA-A*02, HLA-B, HLA-C, and HLA-E gMFI by flow cytometry. Shown are the infected cell average fold changes for each condition from 6 independent cytokine experiments (n=12 donors) and 7 independent co-culture experiments (n=15 donors). Statistical analysis: one sample t test comparison to 1.0, *p<0.05, ***p<0.001, and ****p<0.0001. See also Fig S6F for JR-CSF and REJO.c experiments. (E-G) Effects of TNF-𝘢 pathway manipulation on HLA expression and killing. (E and F) The TNFRSF1A gene was disrupted using CRISPR-Cas9 to ablate TNFR1 expression on CD4 + T cells, followed by HIV infection and overnight cultures with either 1µg/mL recombinant TNF-𝘢 or NK cells (E:T 1). A non-targeting guide RNA was used as a control. Shown are data from 5 independent experiments (n=6 donors). (E) Fold changes in HLA-A*02 and HLA-B expression were measured via flow cytometry. Statistical analysis: multiple paired t tests, **p<0.01 and ***p<0.001. See also Fig S6G and H for IFNGR1 and IFNAR1 knockouts, respectively. (F) NK cell killing of control vs TNFR1 KO infected cells was assessed via flow cytometry. Statistical analysis: paired t test. (E) HIV 89.6 -infected cells were pre-treated overnight with 1µg/mL recombinant TNF-𝘢, followed by overnight co-culture with NK cells (E:T 1) to assess killing. Shown are data from 4 independent experiments (n=8 individual donors). Statistical analysis: paired t test.

Article Snippet: Recombinant cytokines were spiked in at final concentrations of 1μg/mL per cytokine: TNF-α (Biolegend, Cat.#570104), IFN-γ (Biolegend, Cat.#713906), and IFN-β (R&D Systems, Cat.#8499-IF/CF).

Techniques: Infection, Co-Culture Assay, Expressing, Recombinant, Cell Culture, Flow Cytometry, Comparison, CRISPR, Control

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV infection activates the innate immune response. ( A ) Viral load in subcultures harvested at different time points post-infection was quantified by qRT-PCR targeting the viral N gene. All the experiments were performed in triplicate. ( B ) IFN-β levels in PHEV-infected samples were determined by ELISA. Cells infected with VSV (MOI = 1) or treated with Poly(I:C) (20 μM, 24 h) served as positive controls. ( C ) QRT-PCR analysis of IFNA and IFNB1 mRNA expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( D ) QRT-PCR analysis of ISGs (Mx, OAS, GBP, STAT) expression in N2a cells at 24 and 48 h post-PHEV infection. ( E ) QRT-PCR analysis of RIG-I, MDA5, IRF3, and IRF7 expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( F ) WB analysis of RIG-I, IRF7, MAVS, and viral N protein levels in N2a cells at indicated times (0–48 h) after PHEV infection. ( G ) Detection of PHEV, RIG-I, IRF3, IRF7, and IFNB1 in brain tissues from mice at 5 days post-PHEV infection. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: IRF3 initiates the interferon response to suppress viral replication. ( A ) Inhibition of PHEV replication by recombinant IFN-β. WB analysis of viral protein levels to evaluate the suppression of PHEV replication by recombinant IFN-β (0.5 or 1 μg/mL). ( B ) QRT-PCR analysis of IRF3 mRNA in N2a cells overexpressing Myc-tagged IRF3 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( C ) QRT-PCR analysis of IRF7 mRNA in N2a cells overexpressing Myc-tagged IRF7 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( D ) QRT-PCR analysis of PHEV mRNA expression as described in panels B and C . ( E ) WB analysis of PHEV N protein as described in panels B and C . ( F ) QRT-PCR analysis of IFNB1 mRNA expression as described in panels B and C . Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test). ns, not significant.

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Inhibition, Recombinant, Quantitative RT-PCR, Infection, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV N protein targets IRF3 to suppress IFN production. ( A ) Schematic illustration of the experimental design for the dual-luciferase assay using HT1080 cells with a stably integrated IFN-β promoter reporter. ( B ) PHEV N protein inhibits the IFN-β promoter. HT1080 cells were transfected with PHEV proteins, cultured for 24 h, and then stimulated with Poly(I:C) (20 μM) for another 24 h to induce IFN-β promoter activity. IFN-β-Luc reporter activity is normalized to that of Renilla luciferase and shown. Detection of viral protein expression by WB. ( C ) WB analysis demonstrated that the protein levels of RIG-I and IRF3 in N2a cells were unaltered by transfection with a gradient of N protein (0.5–4 μg). ( D ) Nuclear-cytoplasmic fractionation. N2a cells were transfected with 2 μg GFP-N recombinant plasmid for 24 h, and then treated with Poly(I:C) (20 μM) for 24 h or infected with VSV (MOI = 1) for 12 h. Cells were collected for cytoplasmic and nuclear isolation. WB analysis of IRF3 nuclear translocation using cytoplasmic and nuclear fractions prepared from harvested cells. ( E ) N2a cells were infected with PHEV for 24 h (with Poly(I:C) treatment (20 μM, 24 h) as a positive control), followed by immunostaining with anti-N (red) and anti-IRF3 (green) antibodies. Nuclei were counterstained with Hoechst (blue). Scale bar, 10 μm. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: The IFN-β recombinant protein was purchased from MCE.

Techniques: Luciferase, Stable Transfection, Transfection, Cell Culture, Activity Assay, Expressing, Fractionation, Recombinant, Plasmid Preparation, Infection, Isolation, Translocation Assay, Positive Control, Immunostaining, Two Tailed Test