Dataset Information

SINV-nsP3 mScarelt-tagged immunoprecipitation

ABSTRACT: 1x107 cells HEK293 FLP-IN T-REX cells were infected with SINVnsP3-mScarlet (bait) or SINVwt (negative control) at 1 MOI. After 18 hours, cells were scrapped and spin down at 300 g 4°C for 10 mins. After the centrifugation, supernatants were discarded and cell pellet washed once with 1X PBS, followed by another centrifugation at 300xg 4°C for 10min. After discarding the supernatant, cell pellets were lysed with 200µL ice-cold lysis buffer (10mM Tris-Cl pH 7.5, 150mM NaCl, 0.5mM EDTA, 0.5% IGEPAL, benzonase(1U/mL) and 0.1mM AEBSF serine protease inhibitor) , and incubated on ice for 30min. Then, cell lysates were cleared by centrifugation 17,000xg for 10min at 4°C. The supernatants were transferred to new tubes and diluted 1:1 with the dilution buffer (10mM Tris-Cl pH 7.5 150mM NaCl 0.5mM EDTA 0.05% IGEPAL). Next, cell lysates were incubated with 15µL of RFP-Trap® Magnetic Particles M-270 (pre-washed once with the dilution buffer) for 1 hour at 4 °C with mild rotation. After the incubation, the magnetic beads were washed five times with wash buffer1 (10mM Tris/Cl pH 7.5, 150mM NaCl, 0.5mM EDTA and 0.25% IGEPAL), followed by two washes with the dilution buffer. The proteins elution from the beads was done twice at room temperature with shaking, the first elution with 45µL 0.2M glycine pH 2 solution and the second elution with 45µL 0.2M glycine pH 2 (supplemented with 1%SDS) solution. Finally combined protein eluates were neutralized with 10µL 1M Tris-base pH 10. For proteomic analysis three biological replicates were generated.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Wael Kamel

LAB HEAD: Alfredo Castelo

PROVIDER: PXD054117 | Pride | 2024-12-19

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	LUMOS_YD1_WK1_2211014_WK1_02.raw	Raw
	LUMOS_YD1_WK1_2211014_WK2_04.raw	Raw
	LUMOS_YD1_WK1_2211014_WK3_06.raw	Raw
	LUMOS_YD1_WK1_2211014_WK4_08.raw	Raw
	LUMOS_YD1_WK1_2211014_WK5_10.raw	Raw

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Publications

Alphavirus infection triggers selective cytoplasmic translocation of nuclear RBPs with moonlighting antiviral roles.

Kamel Wael W Ruscica Vincenzo V Embarc-Buh Azman A de Laurent Zaydah R ZR Garcia-Moreno Manuel M Demyanenko Yana Y Orton Richard J RJ Noerenberg Marko M Madhusudhan Meghana M Iselin Louisa L Järvelin Aino I AI Hannan Maximilian M Kitano Eduardo E Moore Samantha S Merits Andres A Davis Ilan I Mohammed Shabaz S Castello Alfredo A

Molecular cell 20241211 24

RNA is a central molecule for viruses; however, the interactions that viral RNA (vRNA) establishes with the host cell is only starting to be elucidated. Here, we determine the ribonucleoprotein (RNP) composition of the prototypical arthropod-borne Sindbis virus (SINV). We show that SINV RNAs engage with hundreds of cellular proteins, including a group of nuclear RNA-binding proteins (RBPs) with unknown roles in infection. We demonstrate that these nuclear RBPs are selectively translocated to the ...[more]

PMID: 39642884

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