Enrichment of CB proteins using proximity biotinylation identifies 70 new CB proteins. (A) Schematic of APEX2 biotinylation and experimental pipeline for APEX2 MS experiments used in this study. (B) Immunofluorescent staining of coilin-APEX2 HeLa cell line with construct labeled by α-V5 antibody (magenta) and α-coilin, α-Sm (Y12), or α-Nopp140 antibody (cyan). Scale bars = 10 µm. (C) Immunofluorescent staining of coilin-APEX2, ΔNTD-APEX2, and APEX2-NLS after the addition of biotin phenol (BP) (30 min) and hydrogen peroxide (1 min), cells were stained with α-V5 antibody (magenta) and fluorescent streptavidin to label for biotin (cyan). Scale bars = 10 µm. (D) Normalized biotin intensity measured in CBs, nucleolus and nucleoplasm of Coilin-APEX2 cell line. The biotin intensities are normalized to mean signal in the nucleoplasm. Median of normalized biotin intensity is shown by the cyan line. P values obtained by unpaired t test: ****, P < 0.0001; ns, not significant. (E and F) Label-free quantification (LFQ) of CB proteome identification using coilin-APEX2 relative to APEX2-NLS (E) or relative to ΔNTD-APEX2 (F). Number of replicates: coilin-APEX2 = 4; APEX2-NLS = 4; ΔNTD-APEX2 = 3. Known CB proteins are labeled and colored orange, new significant hits are colored cyan.