Antibody-mediated proximity biotinylation by pA-APEX2 A. Illustration of antibody-mediated pA-APEX2 proximity labeling. pA-APEX2 is recruited to the targeting sites by specific histone modification antibodies. BP and H2O2 are added to cells fixed with 0.1% formaldehyde and incubated for 1 min to induce biotinylation of proteins less than 20 nm adjacent to APEX2. B. Biotinylated proteins are purified using streptavidin beads and analyzed by LC–MS/MS. C. Fluorescence imaging of histone modifications and antibody-mediated biotinylation. H3K9me3 and H3K27me3 were visualized by immunofluorescence staining (green). Biotinylation was induced as indicated before and visualized by staining with streptavidin-Cy3 (red). Nuclei were counterstained with Hoechest33342. Scale bars, 10 μm. D. pA-APEX2-mediated protein labeling in whole-cell lysates. Whole-cell extracts from MEF cells were incubated with pA-APEX2 and H3K27me3 antibody, and biotinylation was induced as indicated before and analyzed by Western blotting. The lower panel shows Ponceau S staining as a loading control. pA, protein A; APEX2, ascorbate peroxidase 2; BP, biotin-phenol; LC–MS/MS, liquid chromatography–tandem mass spectrometry; MEF, mouse embryonic fibroblast; IP, immunoprecipitation.
