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

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.