Fig. 2
Identification of biotinylated 11β-HSD1 by MS from cells stably expressing H6PD-BirA*HA and 11β-HSD1-FLAG. A Scheme of the BioID approach for the detection of potential H6PD interactors. MDA-MB-231 cells were co-transfected with H6PD-BirA*-HA and 11β-HSD1-FLAG, followed by selection of a cell clone with stable expression of the two constructs. Cells stably expressing H6PD-BirA*-HA and 11β-HSD1-FLAG were incubated with 50 µM biotin for 72 h. Biotinylated proteins were then isolated from the cell lysate by affinity purification using streptavidin covalently coupled to magnetic beads and analyzed for the presence of biotinylated proteins by label-free quantitative MS. (Figure created using BioRender.com). B Immunoblotting of lysates from different clones expressing H6PD-BirA*-HA, H6PD-BirA*-HA/11β-HSD1-FLAG or 11β-HSD1-FLAG. Membranes were probed with HRP-labeled streptavidin to detect biotinylated proteins. β-Actin served as a loading control. C H6PD interactome proposed by the BioID approach using H6PD-BirA*-HA and 11β-HSD1-FLAG co-expressing MDA-MB-231 cells. Volcano plot representing the enrichment of peptides from H6PD and 11β-HSD1 in the biotinylated fraction of proteins from MDA-MB-231 cells co-expressing H6PD-BirA*-HA and 11β-HSD1-FLAG compared to parental MDA-MB-231 cells. Proteins were considered enriched if log2-fold change was ≥ 0 and the p-value was ≤ 0.01 (dashed line). Proteins that met this enrichment criteria, including 11β-HSD1, are highlighted by the green box and represent potential H6PD interactors. The x-axis of the presented volcano plot shows the ratio (log2-fold-change) of median protein abundance in samples from MDA-MB-231 cells stably co-expressing H6PD-BirA*-HA and 11β-HSD1-FLAG. The relative intensities of biotinylated peptides derived of D 11β-HSD1 and E) H6PD were determined in the cell clones as indicated. Data represent mean ± SD from three independent experiments. One-way ANOVA with Tukey’s post hoc test, p-values: * < 0.05, ** < 0.01