Journal: Science Advances
Article Title: NDR2 promotes the antiviral immune response via facilitating TRIM25-mediated RIG-I activation in macrophages
Figure Lengend Snippet: NDR2 facilitates RNA virus–induced IFN-β, IL-6, and TNF-α production via a kinase activity–independent mechanism in macrophages. ( A and B ) IFN-β, IL-6, and TNF-α expression in NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV in the indicated times was detected by enzyme-linked immunosorbent assay (ELISA) (A) and real-time polymerase chain reaction (PCR) analysis (B). ( C ) Real-time PCR analysis of IFN-β, IL-6, and TNF-α transcripts in NDR2 f/f and Lysm + NDR2 f/f PMs treated with phosphate-buffered saline (PBS) or with the infection of H1N1, SeV, RSV, EMCV, HSV-1, or MHV68 for 8 hours. ( D ) Fluorescence-activated cell sorting (FACS) analysis of enhanced green fluorescent protein (eGFP) fluorescence intensity in NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV-eGFP. ( E ) VSV-eGFP titers by TCID 50 assay in supernatants of NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV-eGFP for 6 hours. ( F ) NDR2 f/f and Lysm + NDR2 f/f PMs were transfected with empty vector (mock), NDR2, or its kinase-inactive mutants overexpressing plasmids for 36 hours and then infected with VSV, followed by real-time PCR analysis of IFN-β, IL-6, and TNF-α expression. AA, K282A/T442A. ( G ) FACS analysis of RAW264.7 cells stably overexpressing NDR2 or its kinase-inactive mutants infected with VSV-eGFP. Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. ns, no significance. * P
Article Snippet: Enzyme-linked immunosorbent assay The protein levels of murine IFN-β, IL-6, and TNF-α in cell culture supernatants and sera were detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers’ instructions (BioLegend and eBioscience).
Techniques: Activity Assay, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Fluorescence, FACS, Transfection, Plasmid Preparation, Stable Transfection