Figure 6
UL48N targets TRAF6, TRAF3 and STING for deubiqutination and inhibition of interferon synthesis. (a) Schematic representation of TLR, DNA sensing (DAI and cGAS) signaling pathways and ubiquitination of signaling molecules. (b) Luciferase assay was done for IFNβ promoter by co-transfection of TRAF6, TRAF3, IRAK1, IRF7 or STING, with UL48N or UL48NΔDUB expression plasmids as depicted. (c,d) Ubiquitination status of TRAF6, TRAF3 and STING was analyzed in the presence of overexpression of UL48N or UL48NΔDUB in HEK293 cells. Co-transfection of HA-K63Ub, myc-TRAF6, myc-TRAF3 or myc-STING with FLAG-UL48N or FLAG-UL48NΔDUB was done as depicted. (d, top) Immunoprecipitated sample was blotted with anti-HA, anti-myc and anti-FLAG antibody to detect respective proteins, (d, bottom) Density of TRAF6, TRAF3 or STING ubiquitination was calculated and compared for each sample by using ImageJ software. Luc: Luciferase, hpt: hours post transfection, Vec: Empty Vector. Shown results are the representative of three (b) or two (d) independent experiments. Differences were considered statistically significant with a *P-value<0.05, **P-value<0.01 and ***P-value<0.001, ns, non-significant difference (P-value>0.05). See also Supplementary Figure S3a-S3b
