Objectives: To identify the 5' untranslated region of Zika virus (ZIKV 5'UTR) RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site (IRES) located in ZIKV 5'UTR and virus production.
Methods: Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining. Subsequently, liquid chromatography-tandem mass spectrometry (LC-MS/MS), bioinformatics analysis, and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.
Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein. Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag at the N terminus of alkaline phosphatase (AP) for fusion expression. Next, 293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis.
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