a, MEFs expressing ARL13B–mCherry–GECO1.2 were stained with acetylated tubulin. GECO1.2 and mCherry fluorescence overlaps with acetylated tubulin staining. Scale bar: 5 μm; merged channels were offset for clarity. b, Ratio maps of MEFs isolated from ARL13B–mCherry–GECO1.2 mice stimulated with 0.05% DMSO (left) or 400 nM SAG (right) for 24 h. Scale bar: 5 μm. c, Quantification of ciliary GECO1.2/mCherry ratios obtained for MEFs with and without SAG stimulation. Ratio increases from 0.4 ± 0.05 to 0.8 ± 0.2 after SAG stimulation (*P < 0.05; n = 20–30 cilia). d, Example ciliary current measured from MEFs treated with 500 nM SAG (SMO agonist) or with DMSO vehicle (0.05%) in culture for 24–36 h in control conditions and after activation with 10 μM calmidazolium (CMZ). e, Scatter and whisker (±s.d.) plots from cilia show total outward (+100 mV) and inward (−100 mV) current measured for both treatment groups. Averages are indicated by the thick horizontal lines and individual cilium current magnitudes are represented as circles. P values resulting from Student’s t-test comparing treatment groups are indicated (*< 0.05; n = 11 cilia).