Identification of POC5 and Centrin-2 proximity interactome. (A) Biotinylation of the centrosome by V5BirA*-POC5 and V5BirA*-Centrin-2. HEK293T cells stably expressing V5BirA*-POC5 and V5BirA*-Centrin-2 were treated with biotin for 18 h. Cells were then fixed and stained for the protein of interest with V5, biotinylated proteins with fluorescent streptavidin, and centrosome with anti-γ-tubulin antibody. DNA was visualized with DAPI. Scale bar, 10 μm. (B) HEK293T cells stably expressing V5BirA*-POC5 and V5BirA*-Centrin-2 were lysed, and biotinylated proteins were precipitated by streptavidin beads. The initial sample (initial) and immunoprecipitated biotinylated proteins (pulldown) were run on a gel and immunoblotted with fluorescent-coupled streptavidin and V5 antibodies. (C) Representative confocal images of HEK293T::V5BirA*-POC5 and HEK294T::V5BirA*-Centrin-2 centrioles expanded using U-ExM and stained for tubulin (magenta) and V5 (green). Scale bar, 1 μm. (D) POC5 and Centrin-2 proximity interactome map. High-confidence proximity interactors of POC5 and Centrin-2 were determined by using NSAF analysis. The interactome map containing the first 100 proximity interactome of Centrin-2 and all the proximity interactors POC5 was drawn in CytoScape and the shared interactome was visualized in green circles. The circle size corresponds to the fold change. (E) Cellular compartment analysis of the shared proximity interactors of Centrin-2 and POC5. The GO analysis of the shared proximity interactome was determined using DAVID. Source data are available for this figure: SourceData F1.