Fig 1: The subcellular distribution of metabolic enzymes and their putative interactors reveals their nuclear neighborhood.(A and B) Putative interactor signal (biotinylation signal measured using fluorescently labeled streptavidin) in the nuclear region of whole cells (A) or isolated nuclei (B) expressing the bait enzymes. “ni” refers to cells where the expression of the bait enzymes was not induced. (C) Representative immunofluorescence images and quantification of ACO2, OGDH, and IDH3G in the nucleus of mouse embryonic stem cells. The quantification was based on immunofluorescence using antibodies against the endogenous enzymes. “2ary” refers to cells stained only with the secondary, fluorophore-conjugated, antibody. DNA was stained with Hoechst (cyan). Scale bars, 15 µm. (D and E) Representative immunofluorescence images and quantification of OGDH (D) and succinylation (E) in naïve mouse embryonic stem cells and in differentiated counterparts. Scale bars, 10 µm. In (A) to (E), points represent quantification (log2 scale) of individual cells or nuclei, with the population mean and SD calculated for each case. In (B), (D), and (E), the biotinylation, OGDH, and succinylation levels were normalized to Hoechst signal. In (A) to (E), significance was assessed using the Wilcoxon rank sum test. a.u., arbitrary units.
Fig 2: Proximity biotinylation MS reveals core nuclear proteins as putative interactors of IDH1, IDH3G, OGDH, and ACO2.(A) Overview of the identified interactors of each bait enzyme. Heatmap shows fold changes in the abundance of each putative interactor in cells expressing the bait enzyme compared to the parental cells. Top: Significantly enriched GO terms for cellular component (top) and “biological process” (bottom) are shown for each cluster. (B) Subcellular distribution of the putative interactors of each bait enzyme. Nucleus and mitochondria refer to proteins detected only in the nucleus and mitochondria, respectively. Nucleus shared, proteins detected in the nucleus and any other compartment. Mitochondria shared, proteins detected in the mitochondria and any other compartment excluding nucleus. (C) Correlation between the percentage of interacting nuclear proteins and the abundance of the bait enzymes; the latter was estimated using immunofluorescence (main panel) or proteomics (inset). (D) Correlation between the percentage of interacting nuclear proteins and the biotinylation levels in cells expressing the bait enzymes. (E) Selected putative interactors of the bait enzymes. (F) Subcellular distribution of succinylated peptides in U2OS cells quantified by LOPIT. Inset: Enriched GO terms for nuclear proteins with succinylated peptides. ER, endoplasmic reticulum; PM, plasma membrane.
Supplier Page from OriGene Technologies for Aconitase 2 (ACO2) Human shRNA Plasmid Kit (Locus ID 50)