Fig. 5: iAPEX allows specific quantitative primary cilia mapping in NIH/3T3 cells.

a Scheme of cilia-iAPEX proximity labeling workflow for primary cilia proteomics in NIH/3T3 cells. DAAO-dependent proximity labeling was performed by incubating cells with desthiobiotin tyramide (DTBT) and D-Met. As controls, cells were incubated with DTBT and L-Met, or D-Met only. Sample processing according to schematic in Fig. 4a. b, c After proximity labeling, cells were lysed, biotinylated proteins enriched by streptavidin chromatography, and Input and Eluate samples analyzed by SDS-PAGE and western blotting. b DTBT incubation causes background biotinylation (n = 2 independent experiments). Biotin was detected using fluorescently labeled streptavidin, cilia-APEX2 by GFP-specific antibodies. Input 0.063 %, Eluate 1.2 %. c Cilia proteins were specifically isolated after cilia-iAPEX labeling (n = 2). Indicated proteins were detected using specific antibodies. IMCD3 Ift88^-/- cells served as an antibody control. Input 0.063 %, Eluate 8.8 %. d Two-way hierarchical cluster analysis of NIH/3T3 cell cilia-iAPEX proteome. Zoom on clusters highly enriched in cilia proteins. Full cluster analysis shown in Supplementary Fig. 5. e cilia-iAPEX proteomes of IMCD3 and NIH/3T3 cells show distinct overlap with cell specific differences. Venn diagram depicting proteomic overlap of iAPEX-proximity labeled IMCD3 and NIH/3T3 cells against the cilia-APEX2 proteome¹⁷. f, g Validation of primary cilia localization of proteins previously not linked to cilia. f The representative immunofluorescence micrographs illustrate IMCD3 cells stably expressing both cilia-APEX2 (as localization control) and indicated primary cilia candidate proteins C-terminally fused to an ALFA-tag or ALFA-tag fusion alone (for FCAP33) (n = 3). Upon fixation, primary cilia and basal bodies were visualized using antibodies targeting ARL13B or acTub (cilium) and CEP164 or γTub (basal body) respectively, while the proteins of interest were stained with anti-ALFA antibodies. g Micrographs show NIH/3T3 cells transiently transfected with plasmids expressing CKAP5-GFP or FHDC1-FLAG (n = 1). Cells were fixed and primary cilia stained using indicated antibodies. FHDC1-FLAG was detected using anti-FLAG antibody, CKAP5-GFP by fluorescence. Scale bars = 5 µm in all panels.