Extended Data Fig. 2: ISGylation at K23 and K43 is essential for MDA5 activation.
a, ISGylation of endogenous MDA5 in uninfected NHLFs, determined by IP with anti-MDA5 (or an IgG isotype control) and IB with anti-ISG15. b, RT-qPCR analysis of the indicated transcripts in NHLFs that were transfected for 30 h with the indicated siRNAs and then infected with mutEMCV (MOI 0.005) for 16 h in the absence or presence of anti-IFNAR2 (2 µg ml–1). c, Upper panels: Amino acid sequence of the MDA5 CARDs. Lysine (K) residues are highlighted in red. CTD, C-terminal domain. Middle panels: ISGylation of GST-MDA5-2CARD WT or mutants in HEK293T cells that co-expressed V5-ISG15, determined by GST PD and IB with anti-V5. Lower panels: Densitometric analysis of the ISGylation levels of GST-MDA5-2CARD WT and mutants, normalized to protein levels in GST PD. d, ISGylation of FLAG–MDA5 WT and K23R/K43R in transfected MDA5 KO HEK293 cells that co-expressed V5-ISG15 and were stimulated with EMCV-RNA (0.4 µg ml–1) for the indicated times, determined by FLAG PD and IB with anti-V5. e, SUMOylation of GST-MDA5-2CARD WT and K23R/K43R in transfected HEK293T cells that co-expressed Myc-UBE2I and FLAG-SUMO1, determined by FLAG PD and IB with anti-GST. f, Ubiquitination of GST-RIG-I-2CARD WT, or GST-MDA5-2CARD WT and K23R/K43R in HEK293T cells, determined by GST PD and IB with anti-Ub. g, IFN-β-luciferase activity in HEK293T cells transfected for 40 h with GST, or GST-MDA5-2CARD (GST-2CARD) WT or mutants. h, Endogenous IRF3 dimerization in HEK293T cells transfected with GST, or GST-MDA5-2CARD WT or K23R/K43R for 24 h, determined by NativePAGE and IB with anti-IRF3. WCLs were analyzed by SDS-PAGE and IB with anti-GST, anti-IRF3, and anti-actin. i, Validation of MDA5 gene editing in SVGAs. MDA5 protein abundance in WT control and MDA5 KO SVGAs treated with IFN-β (1,000 U ml–1) for 16 h, or left untreated, assessed by IB with anti-MDA5. Immunoblotting for RIG-I and actin served as control. Data represent at least two independent experiments with similar results (mean ± s.d. of n = 3 biological replicates in b and g). **P < 0.01, ***P < 0.001 (two-tailed, unpaired Student’s t-test).
