Journal: Journal of Virology
Article Title: PEDV Nsp14 induces mitophagy-mediated degradation of MAVS to antagonize host innate immunity and facilitate viral proliferation
doi: 10.1128/jvi.00498-25
Figure Lengend Snippet: PEDV Nsp14 recruits NDP52 to mitochondria to induce mitophagy. ( A, B ) IPEC-J2 cells were transfected with the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector for 36 h, followed by IP using protein G magnetic beads pre-incubated with anti-NDP52 or anti-TOM20 pAbs, and subsequent WB analysis. ( C ) IPEC-J2 cells were transfected with the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector for 36 h. The cytoplasmic and mitochondrial fractions were isolated for WB analysis with the indicated antibodies. ( D ) IPEC-J2 cells were transfected with the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector for 36 h. The cells were subjected to confocal microscopy using the specific antibodies and fluorescent reagents. The assessment of co-localization was conducted by calculating the Manders’ overlap coefficient using the JaCoP plugin in the ImageJ software. Scale bars = 10 µm. ( E ) IPEC-J2 cells were infected with or without PEDV for 36 h. The cells were subjected to confocal microscopy using the specific antibodies and fluorescent reagents. The assessment of co-localization was conducted by calculating the Manders’ overlap coefficient using the JaCoP plugin in the ImageJ software. Scale bars = 10 µm. ( F ) IPEC-J2 cells were transfected with siNC or siNDP52. At 24 hpt, the cells were transfected with the pCAGGS-HA-Nsp14 plasmid or HA-tagged empty vector, followed by WB.
Article Snippet: Rabbit anti-TOM20 polyclonal antibodies (pAbs, 11802-1-AP), horseradish peroxidase (HRP)-labeled mouse anti-β-actin monoclonal antibody (mAb, HRP-66009), rabbit anti-NDP52 pAbs (12229-1-AP), and rabbit anti-OPTN pAbs (10837-1-AP) were purchased from Proteintech (Wuhan, China).
Techniques: Transfection, Plasmid Preparation, Magnetic Beads, Incubation, Isolation, Confocal Microscopy, Software, Infection
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