Fig. 1: Background biotinylation in various cell types limits APEX-based proximity labeling applications.
a Immunofluorescence micrographs of IMCD3, C2C12, 3T3-L1, NIH/3T3, and IMCD3 cells stably expressing NPHP31–200-GFP-APEX2 (cilia-APEX2). Cilium formation was induced by 24 h growth factor deprivation. Cells were left untreated (–) or subjected to APEX2 proximity labeling (+) by incubation with 500 µM biotin tyramide (BT) for 30 min followed by 1 mM hydrogen peroxide (H2O2) for 1 min (3T3-L1) or 3 min (remaining cells). After fixation, primary cilia were visualized by anti-ARL13B antibody staining, cilia-APEX2 by GFP fluorescence, and biotin by fluorescently labeled streptavidin. Same imaging parameters for all images. b Wild-type (WT), cilia-APEX2-expressing IMCD3 and NIH/3T3 cells were lysed before (–) or after (+) APEX2 proximity labeling, followed by SDS-PAGE and western blot analysis (n = 4 independent experiments). Biotin detection as in (a), equal protein loading confirmed by total protein stain. c Diagram of cilia-iAPEX expression cassette with NPHP31–200-GFP-APEX2 (cilia-APEX2) and NPHP31–200-FLAG-DAAO (cilia-DAAO) transgenes in head-to-head orientation. A vector containing this cassette allows stable genomic integration via Flp-In recombinase and low-level expression of cilia-APEX2 and cilia-DAAO from truncated cytomegalovirus promoter (pCMVΔ6) and EF1α promoter lacking the TATA box (pEF1αΔ), respectively107. Enzymes are fused to N-terminal cilia-targeting sequences (amino acids 1-200 of murine Nephrocystin-3 (mNphp3)) and tagged with enhanced GFP (EGFP) for APEX2 and FLAG for DAAO. d Schematic: primary cilium harboring the in situ APEX activation (iAPEX) proximity labeling enzymes. APEX2 and DAAO are genetically targeted to primary cilia using constructs displayed in (c). A D-amino acid (D-AA) serves as DAAO substrate for in situ hydrogen peroxide (H2O2) production through oxidative deamination. Locally produced H2O2 and biotin tyramide (BT) are APEX2 substrates for proximity biotinylation, overcoming the need for external H2O2 addition (red X). e Immunofluorescence micrographs showing APEX2 proximity labeling in primary cilia of IMCD3 cells stably expressing the cilia-targeted iAPEX enzyme cascade (n = 5 independent experiments). APEX2 proximity labeling: incubation with biotin tyramide (BT) for 30 min and H2O2 for 3 min. DAAO-facilitated proximity labeling: D-alanine (D-Ala, 10 mM) added during BT incubation for 30 min. Cilia-DAAO is detected by anti-FLAG antibodies, all others as in (a). All scale bars = 5 µm.
