Figure 2. Ciliary proteins from APEX-labeling in IMCD3 cells.

(A) Venn diagram showing identified proteins after streptavidin chromatography from the indicated APEX-labeling reactions. The number in the yellow segment represents cilia-specific proteins enriched at least 5-fold in the experimental sample compared to both controls.

(B) Venn diagram showing the overlap of cilia-specific proteins between the three independent replicates. The yellow segment represents candidate ciliary proteins identified in at least 2 out of 3 experiments and with a total spectral count ≥6 (Tier 1).

(C) Candidate ciliary proteins from (B) grouped into functional categories. Asterisks denote cilia-specific proteins from Tier 2 that are known interaction partners of Tier 1 proteins. Previously known ciliary proteins are shown in grey, novel candidate ciliary proteins in black. Blue are novel ciliary proteins confirmed by independent methods in this study (see Figures 3, 5 and S2). The complete list of identified proteins is shown in Table S1.

(D) IMCD3 cells were transiently transfected with the indicated fusion proteins for 24 h, after which ciliation was induced for another 24 h before cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100 and proteins detected by GFP fluorescence (LAP-SKA1), anti-FLAG or anti-HA antibodies. Cilia were counterstained using anti-ARL13B or anti-acTub antibodies as indicated. Only merged images are shown (see Figure S2 for individual channels). Merged insets show primary cilia with channels shifted to aid visualization. Scale bars represent 5 μm (main panels) and 1 μm (insets).

(E) Table comparing prominent protein categories identified in primary cilia by PCP (Ishikawa et al., 2012) vs. cilia-APEX. Note the absence of ribosomal subunits and chaperones, frequent contamination of proteomic studies in the cilia-APEX proteome. See also Figure S2.