Journal: Frontiers in microbiology
Article Title: Synthetic mimetics assigned a major role to IFNAR2 in type I interferon signaling.
doi: 10.3389/fmicb.2022.947169
Figure Lengend Snippet: FIGURE 2 Comparable pattern and intensities of STAT activation by native and synthetic type I IFN receptors. (A) Whereas native type I IFN signaling depends on IFNAR1/2 heterodimers, the synthetic cytokines can engage different IFNAR1/2 receptor combinations. IFNAR1 (myc-tagged, lagoon) and IFNAR2 (HA-tagged, blue) which are associated with Tyk2 and Jak1, respectively. In the synthetic receptors, the extracellular domains of IFNAR1 and IFNAR2 were replaced by nanobodies directed against GFP (green) and mCherry (red), respectively. Native IFNARs are activated by type I IFNs, synthetic IFNARs were activated by multimeric GFP/mCherry ligands (GC, GG, CC, GCCG). (B) STAT1 and STAT2 activation in HEK293 cells transiently expressing murine IFNAR1 and IFNAR2 (left), murine VGIFNAR1 and VCIFNAR2 (middle) or human VGIFNAR1 and VCIFNAR2 (right) treated with 1,000 U/ml IFNα4, 10 ng/ml Hyper-IL-6 or 100 ng/ml of the synthetic cytokine ligands (GCCG, GG, CC, GC) for 30 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT1, phospho-STAT2 and STAT1, STAT2, IFNAR1 (myc-tagged), and IFNAR2 (HA-tagged). Western blot data shows one representative experiment out of three. (C) STAT1 activation in Ba/F3-gp130 cells after stimulation with 200 U/ml IFNα4, 100 ng/ml IFNβ, 20 ng/ml IFNγ, 20 ng/ml IFNλ2, 10 ng/ml Hyper-IL-6, or 100 ng/ml of the synthetic cytokine ligands GC for 30 min. Equal amounts of proteins (50 μg/lane) were analyzed via specific antibodies detecting phospho-STAT1 and STAT1. Western blot data shows one representative experiment out of three. (D) Cell surface expression of Ba/F3-gp130 cells transfected with either murine or human VGIFNAR1 and VCIFNAR2. Expression was detected via antibodies detecting myc-tagged IFNAR1 (upper panel) or HA-tagged IFNAR2 (lower panel)
Article Snippet: Recombinant murine IFNβ [#8234-MB-010/CF), IFNγ (#485-MI-100) and IFNλ2 (#4635-ML-025) were purchased from R&D Systems (bio-techne, Minneapolis, MN, United States).
Techniques: Activation Assay, Expressing, Western Blot, Transfection
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