Fig. 2.

TurboID biotinylates intracellular and secreted proteins in pericyte spheroid cultures.A, illustration of the experimental workflow and cells used for spheroid cultures. B, cryosection of spheroid immune-stained for PDGFRβ (pericytes), CD31 (endothelial cells), and GFAP (astrocytes). C, spheroids with TurboID-pericytes treated with 500 μM biotin for 24 h or negative controls including TurboID-pericytes with omitted biotin or WT-pericytes with omitted TurboID ligase were stained for anti-V5 verifying TurboID expression, calnexin marking the ER, and streptavidin-488 for biotin-labeling. D, Western blot of supernatant samples from spheroids using SA-HRP after 24 h of either normoxic (N) or hypoxic (H) incubation. E, total protein content from the supernatant samples from spheroids were analyzed with silver stain. Scale bar represents 50 μm. ER, endoplasmic reticulum; SA-HRP, streptavidin-horse radish peroxidase; TID, TurboID.