Journal: mBio
Article Title: DDX4 enhances antiviral activity of type I interferon by disrupting interaction of USP7/SOCS1 and promoting degradation of SOCS1
doi: 10.1128/mbio.03213-23
Figure Lengend Snippet: DDX4 enhances the IFN-I mediated signaling pathway. ( A ) RT-qPCR analysis of Ifnβ mRNA levels in RAW264.7 (left) and MEF (right) cells transfected with increasing amounts of Myc-DDX4 and then infected with VSV (MOI = 1.0, 10 h). ( B ) RT-qPCR analysis of Ifit1 mRNA levels in Ddx4 +/+ or Ddx4 -/- RAW264.7 cells treated with mIFNβ (300 IU/mL) for 8 h. ( C ) RT-qPCR analysis of IFIT1 mRNA levels in HEK293T cells transfected with empty vectors or Myc-DDX4 and then treated with IFNα (1,000 IU/mL). ( D ) Western blot analysis of PKR and IFIT1 proteins in Ddx4 +/+ or Ddx4 -/- RAW264.7 cells treated with mIFNβ (300 IU/mL) for the indicated times. ( E ) Western blot analysis of PKR and IFIT1 protein in HEK293T cells transfected with empty vectors or Myc-DDX4 and then treated with IFNα (1,000 IU/mL) for the indicated times. ( F ) Western blot analysis of PKR and IFIT1 protein in HEK293T cells transfected with increasing amounts of Myc-DDX4 and then treated with IFNα (1,000 IU/mL) for 24 h. ( G ) Western blot analysis of p-STAT1 in Ddx4 +/+ or Ddx4 -/- RAW264.7 cells treated with mIFNβ (300 IU/mL) for the indicated times. ( H ) Western blot analysis of p-STAT1 in HEK293T cells transfected with empty vectors or Myc-DDX4 and then treated with IFNα (1,000 IU/mL) for the indicated times. ( I ) Fluorescence microscopy of VSV-GFP viruses and VSV-GFP fluorescence intensity in HEK293T cells transfected with empty vectors or Myc-DDX4 and then treated with IFNα (50 IU/mL) for 20 h, then infected with VSV-GFP (MOI = 1) for 24 h. Scale bars, 100 µm. Data are shown as mean ± SD of three biological replicates ( A, B, and C ) or are representative of three independent experiments ( D–H ). NS, not significant; ** P < 0.01 and *** P < 0.001.
Article Snippet: The antibodies with indicated dilutions were as follows: DDX4 (Affinity, AF4098, 1:500), VSV-G (Santa Cruz, sc-66180, 1:2,000), PKR (Cell Signaling Technology, sc-6282, 1:1,000), IFIT1 (Santa Cruz, sc-134948, 1:1,000), GFP (Santa Cruz, sc-9996, 1:3,000), p-STAT1 (Cell Signaling Technology, 9617, 1:1,000), STAT1 (Cell Signaling Technology, 8826, 1:5,000), HA (Sigma, ab9110, 1:5,000), Myc (Abmart, M20002, 1:5,000), Ub (Santa Cruz, sc-8017, 1:500), USP7 (Santa Cruz Biotechnology, sc-30164, 1:1000), SOCS1 (Millipore, Billierica, MA; 04–002, 1:1,000), IFNAR1 (Abcom, ab45172, 1:1,000), and Tubulin (Proteintech, 66031–1-Ig, 1:5,000).
Techniques: Quantitative RT-PCR, Transfection, Infection, Western Blot, Fluorescence, Microscopy
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