Skip to main content
. Author manuscript; available in PMC: 2015 Jun 19.
Published in final edited form as: Immunity. 2014 Jun 12;40(6):936–948. doi: 10.1016/j.immuni.2014.05.007

Fig. 4. OASL enhances RIG-I signaling.

Fig. 4

(A) OASL expression enhances IFNβ-reporter activity. HEK293 cells were co-transfected either with control pcDNA3 (1), pcDNA3-OAS1 (2) or pcDNA3-OASL (3) in combination with IFNβ-luciferase reporter and β-actin Renilla luciferase reporter for 24 h. One part of the transfected cells was used for IB to detect protein expression (inset) and the other part was stimulated with SeV for 16 h and luciferase activities were measured. (B–C) OASL expression enhances RIG-I signaling. Cells were infected with SeV for 16 h followed by IB using ISG56, ISG60, V5 (OASL) and actin antibodies (B). Cells were transfected with LMW for 24 h followed by IB using indicated antibodies (C). (D–E) IRF3 dimerization and nuclear localization is enhanced in OASL expressing cells. Indicated cells were stimulated with SeV for 12h. Cell lysates were analyzed in native-PAGE followed by IB with IRF3 antibody (D). Nuclear extracts (NE) from similarly stimulated cells were analyzed along with control uninfected cytoplasm (CP) by IB for IRF3 and nuclear marker DRBP76 respectively (E). (F) NF-κB-dependent gene inductions are also enhanced in OASL expressing cells. Indicated cells were either transfected with LMW or infected with SeV for 24 h followed by qRT-PCR analysis using indicated primers to detect NF-κB-controlled gene induction. (G) Dose-dependent enhancement of RIG-I-signaling sensitivity by OASL. HT1080-iOASL cells were stimulated with Doxycycline at indicated concentrations for 48h followed by infection with SeV (2 HAU/ml) for 8 h. Cell lysates were analyzed by IB as indicated. (H) UBL deleted OASL mutant does not enhance SeV mediated IFNβ induction. Cells were either mock infected or infected with 20 HAU/ml SeV for 16 h. Total RNA from each well were extracted followed by qRT-PCR using IFNβ specific primers. (I) OASL mediated enhancement of ISG induction by SeV is dependent on its UBL. Various OASL expressing HT1080 cells were infected with SeV as indicated for 16 h followed by IB with indicated antibodies as before.