Rer1p knockdown in S12 cells decreases Hh signaling. (a) Gli luciferase reporter assay reveals that Rer1p depletion inhibits Hh signaling similarly to Ift88 knockdown but less than depletion of Smoothened (Smo) or luciferase (Luc); Rer1F is a FAM-labeled Rer1 siRNA. NSC, nonspecific control. (b, left) Smoothened is transported normally to the cilia of Rer1p-deficient cells after 21-h Hh stimulation. (insets) Magnification of boxed areas with the red Smoothened channel displaced six pixels to the right. Arrows point to tips of cilia: white for Smoothened⁻ and yellow for Smoothened⁺ cilia. (right) Quantification of Smoothened translocation to cilia. (c, left) Rer1p inhibits ciliary accumulation of Gli3 (red). Arrows point to cilia tips: white for Gli3⁻ and yellow for Gli3⁺ cilia. (right) Quantification of Gli3 accumulation at cilia tips. (b and c, insets) Bars, 3 µm. (d) Hh-dependent Gli3 degradation, but not processing, is inhibited by Rer1 depletion. (e) Representative Western blot and quantification (n = 4 independent experiments) of Gli3FL and Gli3R normalized to tubulin. Acetylated and γ-tubulin are shown in green, and DAPI is shown in blue (b and c). Represented are means ± SD of more than three experiments. *, P ≤ 0.05; **, P ≤ 0.01 by Student’s t test.