开发一种生物素化tgn常驻蛋白的方法。(A)内源性tgn -常驻蛋白的HRP-TGN46生物素化标记示意图。在生物素-苯酚和过氧化氢的存在下,HRP催化形成膜不透水的生物素-phenoxyl,共价标记附近的内源性蛋白质。(B)表达hrp - tgn46的HeLa细胞用DAB和H2O2孵制10分钟的透射电镜图(比尺棒,500 nm;缩放比例尺,100 nm。)ER,内质网;GA,高尔基体;米,线粒体;N,核;NE,核膜。 (C) STED microscopy of HRP-TGN46–expressing HeLa cells following biotinylation, stained with fluorescent streptavidin and immunofluorescence labeling of Golgin-97. (Scale bars, 5 µm; Insets, 2 µm.) (D) Streptavidin affinity isolation of biotinylated proteins from total cell lysate of HRP-TGN46–expressing cells incubated with H2O2 in the presence or absence of BP, and their corresponding known subcellular localization. (E) Scatterplot of all HRP-TGN46–labeled proteins, with known mannose-6-phosphate–tagged proteins overlaid in red, and example TGN retrograde cargoes highlighted by blue labels; n = 5 independent repeats. (F–G) Graphical representation of overrepresentation of gene ontology cellular compartments (F) and biological processes (G) within the HRP-TGN46–proteome according to their calculated P value. Overrepresented gene ontology terms are colored green, and underrepresented terms are colored red. The numbers of identified proteins belonging to each category are provided within parentheses. Credit:美国国家科学院院刊(2022)。DOI: 10.1073 / pnas.2201980119