Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: IFN-α– but not IFN-λ–induced STAT1 phosphorylation becomes refractory to continuous stimulation. A, liver biopsies from chronic hepatitis C patients (n = 16) were divided into three groups based on their IFNL4 genotype (rs368234815; TT/TT, TT/dG, and dG/dG). Total RNA from biopsies and Huh7 and Huh7 LR cells were prepared. Expression of the IFNLR1 transcript was analyzed by quantitative PCR. Results (mean ± S.D.) are shown as copy numbers per 40 ng of total RNA. B and C, Huh7 LR cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for the indicated times. B, p-STAT1, STAT1, p-STAT2, STAT2, p-STAT3, STAT3, USP18, SOCS1, SOCS3, and actin were visualized using specific antibodies. Shown are representative blots from two independent experiments. C, transcripts of interferon-stimulated genes (RSAD2, IFI27, and GBP5) were quantified by PCR. Results (mean ± S.D., n = 3) are shown as relative expression to GAPDH. ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: Overexpression of SOCS1, SOCS3, and USP18 leads to a reduction of IFN-α– and IFN-λ–mediated STAT1 phosphorylation. A, Huh7 LR cells were transiently transfected with control, SOCS1, SOCS3, or USP18 expression plasmids. 24 h later, cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for 30 min, and p-STAT1, STAT1, SOCS1, SOCS3, USP18, and actin were visualized by immunoblotting. Shown are representative blots from three independent experiments. B, Huh7 LR cells were transfected with SOCS1, SOCS3, or USP18 expression plasmids for 24 h. The mRNA expression levels of SOCS1, SOCS3, and USP18 were analyzed by quantitative PCR and compared with the endogenously induced SOCS1, SOCS3, or USP18 upon IFN-α or IFN-λ1 stimulation at the indicated time points. The results (mean ± S.D., n = 3) are shown as relative expression to GAPDH. Protein levels of SOCS1, SOCS3, and USP18 and actin were visualized using specific antibodies. ox, overexpression; ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Over Expression, Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: SOCS1 is a modulator of IFN-λ-signaling. Control, SOCS1−/−, SOCS3−/−, and USP18−/− cells were transfected with pGL3-ISRE-Mx1-Luc and pGL4-CMV-Renilla-Luc plasmids and, 20 h later, stimulated with 100 ng/ml IFN-λ1, 50 ng/ml IFN-λ4, or 1000 IU/ml IFN-α for 4 h, 8 h, and 24 h or left untreated. The firefly luciferase values were normalized to Renilla luciferase, and the results (mean ± S.D., n = 2) are expressed as firefly/Renilla ratio. Unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Transfection, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: SOCS1 is a modulator of IFN-λ–induced ISGs expression in vitro. Control, SOCS1−/−, SOCS3−/−, and USP18−/− cells were stimulated with 1000 IU/ml IFN-α or 100 ng/ml IFN-λ1 for 4 h, 8 h, and 24 h or left untreated, and the expression levels of RSAD2, GBP5, and IFI27 were analyzed by quantitative PCR. The results (mean ± S.D., n = 2) are shown as relative expression to GAPDH. Unpaired t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, In Vitro, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: Depletion of Socs-1 increased IFN-λ–induced ISGs expression in vivo. Control and Socs1−/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2, Oas1, Stat1, Usp18, and Socs1 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19. Three to four animals were used per time point and condition. Unpaired t test with Welch's correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, In Vivo, Injection, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: SOCS1 is an inducible negative regulator of interferon λ (IFN-λ)–induced gene expression in vivo

doi: 10.1074/jbc.M117.788877

Figure Lengend Snippet: Depletion of Usp18 increased IFN-α–induced ISGs expression in vivo. Control and Usp18−/− mice were subcutaneously injected with PBS, 1000 units/g mouse IFN-α, or 50 ng/g mouse IFN-λ2. The liver, the lung, and the gut were collected 4 h and 8 h after injection, and total RNA was prepared. The expression of Rsad2, Oas1, Stat1, Socs1, and Usp18 was measured by quantitative PCR. The results (mean ± S.D.) are shown as relative expression to Rpl19. Three to five animals were used per time point and condition. Unpaired t test with Welch's correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ut, untreated.

Article Snippet: The pCMV6 plasmid containing the SOCS1 gene (RC220847) was purchased from Origene Technologies Inc. (Rockville, MD) and used to overexpress SOCS1.

Techniques: Expressing, In Vivo, Injection, Real-time Polymerase Chain Reaction