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Phosphorylated UPF1 immunoprecipitation in human embryonic kidney 293T cells

ABSTRACT: HEK293T cells were cultured for 3hr in okadaic acid (200 nM; LC Laboratories) and subsequently collected and lysed using Hypotonic Gentle Lysis Buffer with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Cellular p-UPF1 was immunoprecipitated using the rabbit polyclonal anti-p-UFP1 S1116 antibody (Millipore) in the presence of 1 U/ml of RNase I (Thermo Fisher Scientific Ambion). Immunoprecipitated material was electrophoresed in 12% SDS-polyacrylamide, and silver-stained bands using SilverQuest Staining Kit (Thermo Fisher Scientific Invitrogen) were excised and subjected to in-gel trypsin digestion. Chromatographed peptides were identified at the Massachusetts Institute of Technology Whitehead Institute Proteomics Core Facility (http://massspec.wi.mit.edu/) using an Orbitrap EliteTM Hybrid Ion Trap-Orbitap Mass Spectrometer with Dionex Ultimate 3000 Rapid Separation Liquid Chromatography (Thermo Fisher Scientific) and Scaffold Software (version Scaffold_4.2.1, Proteome Software Inc.).

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Lynne Maquat

PROVIDER: PXD014901 | JPOST Repository | Mon Aug 03 00:00:00 BST 2020

REPOSITORIES: jPOST

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			Action	DRS
	orb131126_03_tatsuaki-NC1.raw	Raw
	orb131126_04_tatsuaki-NC2.raw	Raw
	orb131126_05_tatsuaki-NC3.raw	Raw
	orb131126_06_tatsuaki-NC4.raw	Raw
	orb131126_07_tatsuaki-NC5.raw	Raw

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