Fig. 1: BioID workflow for identification of Bid vicinal proteins in mitotic cells. | Cell Death & Disease

Fig. 1: BioID workflow for identification of Bid vicinal proteins in mitotic cells.

From: BioID-based proteomic analysis of the Bid interactome identifies novel proteins involved in cell-cycle-dependent apoptotic priming

Fig. 1

A Schematic diagram of BioID labelling strategy. Selective biotinylation of proximal proteins is followed by stringent cell lysis for streptavidin-affinity purification and identification/quantification by LC–MS/MS. B HeLa cells, either wild type or stably expressing mBidWT–BirA*, mBidS66A–BirA*, mBidG94E–BirA* or venus BirA*, were grown with 50 µM biotin for 16 h in the presence (+) or absence (−) of nocodazole. Whole-cell lysates were prepared and examined by immunoblotting for the indicated antibodies and streptavidin. C Single-cell-fate profiles of HeLa cells in the presence or absence of nocodazole, imaged over 48 h. Each individual horizontal line represents a single cell. Data represent 90 cells tracked over three independent repeats. A biotin-labelling window of 16 h significantly enriched for mitotic cells compared to untreated controls, without significant enrichment for apoptotic cells. D Volcano plot of mean- fold change of biotinylated protein abundance for mBidWT–BirA* vs. venus-BirA* control for unsynchronised and nocodazole-treated samples. Positive ratio indicates enrichment in BidWT sample. P value calculated via ANOVA from three independent replicates.

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