Skip to main content
. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Nat Microbiol. 2021 Mar 16;6(4):467–478. doi: 10.1038/s41564-021-00884-1

Extended Data Fig. 3. Dephosphorylation of MDA5 induces ISGylation.

Extended Data Fig. 3

(a) ISGylation of GST-MDA5–2CARD in HEK293T cells transfected with V5-ISG15 and the indicated siRNAs for 48 h, assessed by GST-PD and IB with anti-V5 and anti-GST. WCLs were probed by IB with anti-V5, anti-PP1α, anti-PP1γ, and anti-Actin. (b) ISGylation of GST-MDA5–2CARD WT or S88A, S88D and S88E mutants in transiently transfected HEK293T cells that also co-expressed V5-ISG15, determined by GST-PD and IB with anti-V5 and anti-GST forty hours after transfection. (c) Phosphorylation of FLAG-MDA5 WT or K23R/K43R mutant in HEK293T cells, determined by FLAG-PD and IB with anti-pS88-MDA5 and anti-FLAG. WCLs were probed by IB with anti-FLAG and anti-Actin. (d) ISGylation and phosphorylation of GST-MDA5–2CARD in HEK293T cells transfected with V5-ISG15 and either empty vector or increasing amounts of FLAG-MeV-V for 24 h, determined by GST-PD and IB with anti-pS88-MDA5, anti-V5, and anti-GST. (e) ISGylation and phosphorylation of FLAG-tagged MDA5 in transfected HEK293T cells that co-expressed V5-ISG15, HA-Ube1L, and FLAG-UbcH8 as well as either empty vector or increasing amounts of HA-MeV-V for 24 h, determined by FLAG-PD and IB with anti-pS88-MDA5, anti-V5, and anti-FLAG. WCLs were probed by IB with the indicated antibodies. (f) ISGylation of GST-MDA5–2CARD, or GST (negative control), in HEK293T cells transiently transfected with V5-ISG15 and either empty vector or HA-tagged MeV-V WT or Δtail for 48 h, determined by GST-PD and IB with anti-V5 and anti-GST. (g) Upper panel: Phosphorylation of HA-MDA5 in transfected HEK293T cells that co-expressed empty vector or the indicated FLAG-tagged paramyxoviral V proteins, assessed by HA-PD and IB with anti-pS88-MDA5 and anti-HA. WCLs were probed by IB with anti-FLAG. Lower panel: Densitometric analysis of the levels of MDA5 S88 phosphorylation, normalized to protein levels in HA-PD. (h) ISGylation and phosphorylation of GST-MDA5–2CARD in transfected HEK293T cells that co-expressed V5-ISG15 and either empty vector or increasing amounts of FLAG-NiV-V or FLAG-HeV-V for 24 h, determined by GST PD and IB with anti-V5, anti-pS88-MDA5, and anti-GST. WCLs were probed by IB with anti-FLAG, anti-V5, and anti-Actin. Data are representative of at least two independent experiments with similar results.