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OASL binds to dsRNA and dsRNA binding is essential to enhance RIG-I signaling. ( A ) A constant concentration of OLD was incubated with increasing amounts of 18 bp dsRNA and subsequently analyzed by silver stained native PAGE. OLD was kept constant at 2.4 μM (1 μg) and incubated with 0, 0.13, 0.27, 0.54, 1.1, 2.2, 4.3 or 8.6 μM (1 μg) 18 bp dsRNA. The negative control was performed with 8.6 μM 18 bp dsRNA and no OLD. ( B ) 2.4 μM wild-type OLD or K66E OLD was incubated with 8.6 μM 18 bp dsRNA and analyzed by native PAGE. The proteins were visualized by silver staining or immunoblotting against the 6xHis-tag. Wells: the position of the bottom of the wells in the corresponding native gel. ( C ) A constant concentration of 18 bp dsRNA was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of 18 bp dsRNA was kept constant at 10 μM and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( D ) A constant concentration of <t>poly(I:C)-LMW</t> was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of poly(I:C)-LMW was kept constant at 0.2 mg/ml and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( E ) HEK293T OASL neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL R45E/K66E/R196E/K200E (RKRK), OASL K63E, OASL K66E or OASL S327A in combination with an IFN-β promoter luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. Ns: P -value > 0.05. ***: P -value ≤ 0.001.
Poly I C, supplied by Cytiva Europe, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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OASL binds to dsRNA and dsRNA binding is essential to enhance RIG-I signaling. ( A ) A constant concentration of OLD was incubated with increasing amounts of 18 bp dsRNA and subsequently analyzed by silver stained native PAGE. OLD was kept constant at 2.4 μM (1 μg) and incubated with 0, 0.13, 0.27, 0.54, 1.1, 2.2, 4.3 or 8.6 μM (1 μg) 18 bp dsRNA. The negative control was performed with 8.6 μM 18 bp dsRNA and no OLD. ( B ) 2.4 μM wild-type OLD or K66E OLD was incubated with 8.6 μM 18 bp dsRNA and analyzed by native PAGE. The proteins were visualized by silver staining or immunoblotting against the 6xHis-tag. Wells: the position of the bottom of the wells in the corresponding native gel. ( C ) A constant concentration of 18 bp dsRNA was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of 18 bp dsRNA was kept constant at 10 μM and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( D ) A constant concentration of poly(I:C)-LMW was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of poly(I:C)-LMW was kept constant at 0.2 mg/ml and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( E ) HEK293T OASL neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL R45E/K66E/R196E/K200E (RKRK), OASL K63E, OASL K66E or OASL S327A in combination with an IFN-β promoter luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. Ns: P -value > 0.05. ***: P -value ≤ 0.001.

Journal: Nucleic Acids Research

Article Title: Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling

doi: 10.1093/nar/gkv389

Figure Lengend Snippet: OASL binds to dsRNA and dsRNA binding is essential to enhance RIG-I signaling. ( A ) A constant concentration of OLD was incubated with increasing amounts of 18 bp dsRNA and subsequently analyzed by silver stained native PAGE. OLD was kept constant at 2.4 μM (1 μg) and incubated with 0, 0.13, 0.27, 0.54, 1.1, 2.2, 4.3 or 8.6 μM (1 μg) 18 bp dsRNA. The negative control was performed with 8.6 μM 18 bp dsRNA and no OLD. ( B ) 2.4 μM wild-type OLD or K66E OLD was incubated with 8.6 μM 18 bp dsRNA and analyzed by native PAGE. The proteins were visualized by silver staining or immunoblotting against the 6xHis-tag. Wells: the position of the bottom of the wells in the corresponding native gel. ( C ) A constant concentration of 18 bp dsRNA was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of 18 bp dsRNA was kept constant at 10 μM and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( D ) A constant concentration of poly(I:C)-LMW was incubated with increasing amounts of either wild-type OLD or the K66E mutant and subsequently analyzed by native PAGE. The concentration of poly(I:C)-LMW was kept constant at 0.2 mg/ml and incubated with 0, 7, 28 or 56 μM OLD wt or K66E. The nucleic acid was visualized by ethidium bromide staining. ( E ) HEK293T OASL neg cells were co-transfected with V5-tagged empty pcDNA3.1 vector (mock), OASL, OASL R45E/K66E/R196E/K200E (RKRK), OASL K63E, OASL K66E or OASL S327A in combination with an IFN-β promoter luciferase reporter construct for 24 h. The cells were either infected with SeV or mock infected for 16 h and the luciferase activities were measured. Error bars indicate standard deviation from three independent experiments. The inset shows an immunoblot against V5 and GAPDH to confirm the expression of the transfected constructs and to ensure that similar amounts were loaded on the gel, respectively. Ns: P -value > 0.05. ***: P -value ≤ 0.001.

Article Snippet: The catalytic activity of the different OAS proteins were assayed in 20 mM HEPES pH 7.5, 10 mM MgCl 2 , 10 mM MnCl 2 , 0.2 mM DTT, 5% (v/v) glycerol, 0.1 mg/ml bovine serum albumin, 0.2 mM EDTA, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM TTP, 0.5 mM UTP and 100 μg/ml poly(I:C) (synthetic dsRNA analog; GE Life Sciences) or 100 μg/ml salmon sperm dsDNA (Invitrogen).

Techniques: Binding Assay, Concentration Assay, Incubation, Staining, Clear Native PAGE, Negative Control, Silver Staining, Western Blot, Mutagenesis, Transfection, Plasmid Preparation, Luciferase, Construct, Infection, Standard Deviation, Expressing