Supplementary Figure 7: Infection-specific EPRS phosphorylation is essential for regulating MAVS.
From: Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity
(a,b) In vitro binding assay showing MAVS interaction with PCBP2 KH1 (aa 11–82). (c,d) The precipitation (ppt) assays revealed no interaction between PCBP2 and LRS in 293T cells (c), whereas PCBP2 interacted with MAVS (d). (e) Ubiquitination of exogenous MAVS in non-targeting control (siCtrl) or EPRS-deficient (siEPRS) 293T cells transfected with Ub, ITCH, MAVS, or PCBP2. (f) Ubiquitination of endogenous MAVS in 293T cells transfected with Ub, ITCH, PCBP2, and Strep-empty vector (EV), or with WT EPRS or its mutant (EP-MT, enzymatically inactive for both tRNA glutamylation and prolylation). (g) Expression of endogenous MAVS in 293T cells transfected with PCBP2 and WT EPRS or EP-MT. The histogram shows the intensity of the MAVS band normalized against actin. (h–n) Ubiquitination of endogenous MAVS (h) in non-targeting control (sgCtrl) or sgEPRS 293T cells transfected with HA-Ub and infected with VSV-GFP (MOI = 0.1). (i) Ubiquitination of endogenous MAVS in 293T cells transfected with HA-Ub and infected with VSV-GFP. (j) IFN-β or (k) IL-6 levels in supernatants from cells infected with VSV-GFP. (l) Fluorescence microscopy images and (m) plaque assay at 24 h post-infection with VSV-GFP (MOI = 0.0001). (n) Immunoblot analysis of EPRS-FLAG or endogenous EPRS. sgEPRS 293T cells were reconstituted with a FLAG-EV, WT EPRS, S990A, or S990D (i–n). Scale bar, 100 μm (l). *P < 0.01; NS, not significant (Student’s t-test; j,k,m). Data are representative of two (a-n) independent biological replicates with similar results (mean and s.d. of triplicate in j,k,m).
