a–l, The first column shows ARL13B–EGFPtg (green) fluorescence; second column labels ACIII (b, e) or acetylated tubulin (h, k). Third column: merged red and green channels were offset for clarity. Primary MEFs of Arl13b-EGFPtg (a–c) and wild-type (d–f) mice isolated from E14.5 embryos. ARL13B–EGFP co-localizes with ciliary ACIII in c. MEFs isolated from wild-type mice show no fluorescence in the cilium (488 nm excitation). g–i, Primary RPE cells isolated from P12 Arl13b-EGFPtg mice. Cells were fixed and stained with antibody to acetylated tubulin. ARL13B–EGFP (g) exclusively localized to the primary cilium identified by antibody to acetylated tubulin (h, i). j–l, Stable cell line (hRPE1) expressing SMO–EGFP. After 2 days of serum starvation, SMO–EGFP labelled the primary cilium of hRPE1 cells (j) as indicated by acetylated tubulin labelling (k, l). Scale bars: c, f, i, l, 5 μm. m, n, To determine whether ARL13B–EGFP expression adversely affected ciliogenesis, ciliary length and per cent of cells with cilia were quantified from wild-type and ARL13B–EGFP-expressing MEFs stained by anti-acetylated tubulin. m, Ciliary length was similar in wild-type and ARL13B–EGFP-expressing MEFs (2.6 ± 0.5 μm versus 2.9 ± 0.8 μm, respectively, n = 200). n, The number of cells with cilia was also comparable between wild-type and ARL13B–EGFP-expressing MEFs (60.2% ± 5.1% versus 65.5% ± 7.3%; n = 120).