Fig. 1. Otulin attenuates RIPA pathway.

A IRF3 linear ubiquitination was analyzed in HT1080 cell line transfected with either empty vector (lanes 1, 2, 3 and 4) or HA-Otulin (lane 5) followed by Sendai virus (SeV) infection (lanes 2, 4 and 5) for 24 h (MOI 10). Immunoprecipitation was performed with IRF3 antibody and immunoblotting was done with LUB9 antibody for detection of linear ubiquitination. Right panel shows the total linear ubiquitination of the input. B IRF3 linear ubiquitination was assessed in HT1080 WT and HT1080 Otulin-knockdown (OT-KD) cells post-SeV infection, using LUB9 antibody. C HT1080 Otulin knockdown cells (OT-KD) were reconstituted with either Otulin WT or enzymatically inactive mutant Otulin C129A and IRF3 linear ubiquitination was assessed by LUB9 antibody post SeV infection. D Otulin-IRF3 interaction was assayed by their co-immunoprecipitation from extracts of HT1080 cells that had been infected with SeV for 1 h and 3 h. E HT1080 WT and HT1080 OT-KD cells were either mock or SeV infected and C-PARP and C-caspase 3 levels were measured for assessing apoptotic cell death at the indicated time points. F SeV-C protein levels were assessed post-SeV infection in WT and OT-KD cells as a measure of viral protein synthesis at the indicated time points. G SeV yields were measured after infection of WT and OT-KD cells with SeV for 24 h (MOI 10). Quantification of western blots and statistical analyses are provided in Supplementary Fig. 8 (mean ± SEM, n = 3; ns > 0.05; *P < 0.05; **P < 0.01, ***P < 0.001).