Fig. 3

MS-based identification of proteins that were biotinylated by H6PD-BirA*-HA. A Schematic overview of the approach used to detect potential H6PD interactors in MDA-MB-231 cells expressing H6PD-BirA*-HA. Samples from cells stably expressing H6PD-BirA*-HA were incubated with 50 µM biotin for 72 h, biotinylated proteins were isolated from the lysate by affinity purification using streptavidin magnetic beads and analyzed for the presence of biotinylated proteins using label-free quantitative MS. (Figure created using BioRender.com). B H6PD interactome identified by the BioID approach in H6PD-BirA*-HA expressing MDA-MB-231 cells. In total, 50 proteins were enriched by a log₂-fold change ≥ 3.33 and a p-value ≤ 0.01 in samples from the H6PD-BirA*-HA clone compared to control samples from MDA-MB-231 cells. Proteins that met these enrichment criteria are highlighted by the green box. The H6PD data point is depicted in red. C Subcellular localization of enriched proteins identified in B. The presented data were determined based on the results from three independent experiments. D Protein–protein interactions of the 50 enriched proteins predicted by the STRING database and pathway annotation based on the reactome pathway database. Predictions were made based on curated databases, experimentally determined information, gene neighborhood, co-occurrence or fusion as represented by lines between knots. Empty nodes represent proteins with no known or predicted 3D-structure, filled nodes indicate that 3D-structure is known or predicted. Color of the nodes represent the identified pathways to which the proteins belong to as described in the figure. Transparent nodes represent proteins that were not assigned to any of the listed pathways. Analysis was done with the highest confidence parameter (0.900)