Supplementary Figure 5: Identification of the viral-infection-specific phosphorylation site in EPRS.

(a) Silver-stained Strep-EPRS (indicated by an asterisk) purified by Strep precipitation assay of 293T cells infected or uninfected (–) with PR8-GFP (MOI = 5). EV, Strep-empty vector. (b) MS/MS spectra for a doubly charged EPRS peptide EYIPGQPPLSQSSDSpS*PTR (MH+ = 2125.93, z = 2+) obtained under uninfected (upper) and PR8-infected (lower panel) conditions. The peptides contain the S886 phosphorylation site (marked by an asterisk). (c–e) Extracted ion chromatogram (XIC) of the tryptic digests under uninfected (upper) and infected (lower panel) conditions, corresponding to doubly charged EYIPGQPPLSQSSDSSPTR (MH+ = 2044.96, z = 2+) (c), doubly charged NQGGGLSSSGAGEGQGPK (MH+ = 1586.72, z = 2+) (d), and triply charged KDPSKNQGGGLSSSGAGEGQGPK (MH+ = 2142.02, z = 3+) (e) peptides from non-phosphorylated (left) and phosphorylated (right, marked by an asterisk) EPRS. ND, not detected. (f) Immunoblot analysis of phosphomimetic (S990D) and phosphorylation-resistant (S990A) EPRS against with anti-phospho-EPRS(Ser990) in 293T cells. (g–j) Immunoblot analysis of EPRS Ser990 phosphorylation in RAW264.7 cells infected with PR8-GFP (MOI =1) (g), 293T cells infected with PR8-GFP (MOI = 5) (h) or VSV-GFP (MOI = 0.001) (i), or cells transfected with 2 μg of Poly(I:C) (j). (k,l) Secreted IFN-γ levels from U937 (k) or RAW264.7 (l) cells infected with PR8-GFP or VSV-GFP. Cells treated with IFN-γ (1000 units/ml) were used as a positive control. (m) Immunoblot analysis of Cp expression in PR8-GFP-infected RAW264.7 cells. Data are representative of three (g-m) independent biological replicates with similar results (mean and s.d. of triplicate in k,l).