Proteomics

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Identification of antiviral RNAi regulators, ILF3/DHX9, recruit at ZIKV stem loop B to protect against ZIKV induced microcephaly


ABSTRACT: The stained gel bands are washed three times with ddH2O, followed by the addition of 150μL of destaining solution to remove the stain. After four rounds of water washing, the gel bands are washed twice with 300μL each of 25mM ammonium bicarbonate, 50% acetonitrile, and 100% acetonitrile, respectively, until the gel dehydrates and turns white. Then, 50μL of 10mM DTT solution is added and the gel is reduced in a 56℃ water bath for 30 minutes. Once the temperature drops to room temperature, an equal volume of 50 mM IAMAC solution is added and the gel is alkylated in the dark for 15 minutes. The gel bands are then washed twice again with 300μL each of 25mM ammonium bicarbonate, 50% acetonitrile, and 100% acetonitrile, and dehydrated until the gel turns white. Next, 15 to 20μL of proteomics-grade trypsin at a concentration of 0.01 ug/ul is added. After the gel has fully rehydrated and become transparent on ice, 30 to 40μL of a 50 mM NH4HCO3 solution containing 10% ACN is added to cover the gel. The gel is then digested overnight at 37℃ in a water bath. After enzymatic digestion, the supernatant is transferred to a new EP tube. To the remaining gel piece, 100 µL of extraction solution (67% acetonitrile with 2% formic acid) is added, incubated at 37℃ for 30 minutes, followed by sonication for 15 minutes, centrifugation, and the supernatants are combined and concentrated by drying, ready for mass spectrometry analysis.

ORGANISM(S): Homo Sapiens

SUBMITTER: Xin Chen  

PROVIDER: PXD054904 | iProX | Wed Aug 14 00:00:00 GMT+01:00 2024

REPOSITORIES: iProX

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Publications

Identification of antiviral RNAi regulators, ILF3/DHX9, recruit at ZIKV stem loop B to protect against ZIKV induced microcephaly.

Lei Zhiwei Z   Gu Yu Y   Liu Ying Y   Liu Hailiang H   Lu Xiaohua X   Chen Weijie W   Zhou Lu L   Pan Pan P   Chen Zhuohong Z   Yue Zhaoyang Z   Ruan Jinhui J   Zhu Leqing L   Li Guangqiang G   Xia Xichun X   Yu Yang Y   Dai Jianfeng J   Chen Xin X  

Nature communications 20250226 1


Zika virus (ZIKV) is a member of the Flaviviridae family and causes congenital microcephaly and Guillain-Barré syndrome. Currently, there is a lack of approved vaccines or therapies against ZIKV infection. In this study, we profile vRNA‒host protein interactomes at ZIKV stem‒loop B (SLB) and reveal that interleukin enhancer binding factor 3 (ILF3) and DEAH-box helicase 9 (DHX9) form positive regulators of antiviral RNA inference in undifferentiated human neuroblastoma cells and induced pluripote  ...[more]

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