Journal: Scientific Reports
Article Title: Interferon regulatory factor 3 is a key regulation factor for inducing the expression of SAMHD1 in antiviral innate immunity
doi: 10.1038/srep29665
Figure Lengend Snippet: ( A ) MARC-145 cells and PAMs were infected or mock infected with PRRSV or NDV at an MOI of 1 for 16 h. The levels of SAMHD1 expression, phosphorylation of IRF3, and STAT1, were analyzed using Western blotting. ( B ) HEK293 cells and MARC-145 cells were transfected with psiRNA vector expressing shRNA targeting STAT1 gene for 36 h, then the cells were cultured in selective medium 50–150 μg/mL Zeocin (Life technologies) for 3 days until cell foci were identified. The cells were treated with IFN-α for 12 h. STAT1 and SAMHD1 expression were analyzed using Western blotting. ( C ) HEK293 and MARC-145 cells transfected with STAT1 WT or STAT1 Y701F plasmids were analyzed for SAMHD1 expression at 48 h post-transfection using western blotting. ( D ) THP-1 cells were either non-differentiated or differentiated overnight with 50 ng/ml of PMA, and then treated with 1,000 U/ml human IFN-α for 0–6 h. Nuclear proteins were extracted and the nuclear translocation of STAT1, IRF3, and SAMHD1 expression were detected using Western blotting. PCNA was used as a protein loading control. Expression levels of SAMHD1 compared to β-actin or PCNA are shown. ( E ) THP-1 cells were mock treated or treated with 1,000 U/mL IFN-α for the indicated times. Quantitative RT-PCR was performed using SAMHD1 specific primers and all data was normalized to β-actin (NS, not significant: p > 0.05). Uncropped images of blots are shown in .
Article Snippet: STAT1 WT and STAT1 Y701F plasmids were purchased from Addgene.
Techniques: Infection, Expressing, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, shRNA, Cell Culture, Translocation Assay, Control, Quantitative RT-PCR
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