Fig. 5: Mff phosphorylation impairs mitochondrial antiviral response.
From: MAVS is energized by Mff which senses mitochondrial metabolism via AMPK for acute antiviral immunity
a Scheme of the murine phosphorylation-negative/mimic Mff constructs used in this study. A point mutation was introduced into FLAG-tagged Mff (WT) at S146 as indicated. b Mff KO MEFs stably expressing each Mff construct were analyzed by immunoblotting using anti-FLAG, anti-Mff, and anti-phospho-Mff antibodies. SD: S146D, SE: S146E, SA: S146A. WT MEFs treated with AICAR (AMPK activator: 2 mM) or compound C (AMPK inhibitor: 20 μM) for 2 h were used as controls. c, d qRT-PCR of IFN-β (c) and IL-6 (d) mRNA in Mff KO MEFs stably expressing each Mff construct with or without poly(I:C)-LMW (1 μg/mL) transfection for 8 h. Data represent means ± SEM of three independent experiments (n = 6, P values; two-tailed unpaired t test). e Representative live-cell images of MitoTracker Red (mitochondria, white) in Mff KO MEFs stably expressing each Mff construct. Scale bars: 10 μm (whole) and 2 μm (inset). f Magnified images of MAVS on mitochondria in Mff KO MEFs stably expressing each Mff construct. The cells were transiently transfected with a mitochondrial marker mitoRFP (red) and immunostained with an anti-MAVS antibody (green). Scale bars: 2 μm. g Co-localization ratio between MAVS and mitoRFP in Mff KO MEFs stably expressing each Mff construct. Data represent means ± SEM (vector, SD, SA: n = 22, 20, 16, P values; two-tailed unpaired t test). h Working hypothesis for the energetic regulation of the mitochondrial antiviral response by Mff phosphorylation. Immunoblot analyses and all microscopic analyses were repeated independently more than three times with similar results. Source data are provided as a Source data file.
