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NLRP6 controls PTEN stability by promoting autophagic degradation of p85α to drive tumorigenesis

ABSTRACT: To identify NLRP6-associated proteins in living cells, we applied an APEX2-based labelling method combined with mass spectrometry. In brief, APEX2 was genetically fused to NLRP6. In the presence of hydrogen peroxide, APEX2 catalysed biotin-phenol to become a biotin-phenol radical that covalently bound to NLRP6 neighbouring proteins (<20 nm). The biotinylated proteins were enriched and collected by streptavidin beads. One-dimensional SDS–PAGE followed by liquid chromatography-tandem mass spectrometry (GeLC–MS/MS) was performed to identify the proteins. To detect ubiquitinated p85α, a Flag-tagged p85α plasmid was transfected into cells. The cell lysates were immunoprecipitated with anti-Flag and separated on a 6% SDS–PAGE gel. After in-gel digestion, the resulting peptides were extracted for GeLC–MS/MS analysis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Brain Glioblastoma Multiforme

SUBMITTER: Bowen Li

LAB HEAD: Jun Cui

PROVIDER: PXD037460 | Pride | 2023-07-02

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	NLRP6-APEX2.csv	Csv
	NLRP6-APEX2.raw	Raw
	checksum.txt	Txt
	p85alpha_ubiquitination.msf	Msf
	p85alpha_ubiquitination.raw	Raw

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NLRP6 controls PTEN stability by promoting autophagic degradation of p85α to drive tumorigenesis

Project description:To identify NLRP6-associated proteins in living cells, we applied an APEX2-based labelling method combined with mass spectrometry. In brief, APEX2 was genetically fused to NLRP6. In the presence of hydrogen peroxide, APEX2 catalysed biotin-phenol to become a biotin-phenol radical that covalently bound to NLRP6 neighbouring proteins (<20 nm). The biotinylated proteins were enriched and collected by streptavidin beads. One-dimensional SDS–PAGE followed by liquid chromatography-tandem mass spectrometry (GeLC–MS/MS) was performed to identify the proteins. To detect ubiquitinated p85α, a Flag-tagged p85α plasmid was transfected into cells. The cell lysates were immunoprecipitated with anti-Flag and separated on a 6% SDS–PAGE gel. After in-gel digestion, the resulting peptides were extracted for GeLC–MS/MS analysis.

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