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. 2020 May 20;219(7):e201912104. doi: 10.1083/jcb.201912104

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© 2020 Desai et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure S1. — Related to Fig. 1: Endogenous Smo is trafficked normally in Smo-Flag-Ub–expressing cells. (A) Wild-type MEFs were transfected with PD22 (Smo-Flag) or GP736 (Smo-Flag-Ub) and selected with Bsd. Confluent cells were serum starved and stimulated with SHH-conditioned medium for 24 h before being fixed and stained for Smo or Smo-Ub (Flag; red), cilia (Arl13b; green; arrows), and nuclei (DAPI; blue). Left image of each pair is a three-color composite; right image shows only the red Flag channel. Each image is maximum projection of a three-image stack taken at 0.2-μm intervals. Scale bar is 10 μm and applies to all images in the panel. (B) Presence of Smo or Smo-Ub in cilia was quantitated from the cells described in A. N is 3 replicates. ****, P < 0.0001; ns, not significant by two-way ANOVA. Error bars indicate SD. (C) Wild-type MEFs were transfected with GP736 (Smo-Flag-Ub) and selected with Bsd. Confluent cells were serum starved for 24 h and then stimulated with SAG for 24 h before being fixed and stained for endogenous Smo (red), cilia (Arl13b; green; arrows), and nuclei (DAPI; blue). Left image of each pair is a three-color composite; right image shows only the red Smo channel. Each image is maximum projection of a three-image stack taken at 0.2-μm intervals. Scale bar is 10 μm and applies to all images in the panel. 3.67% ± 3.2% of cells were Smo positive without SAG stimulation, and 92.7% ± 3.2% of cells were Smo positive after SAG stimulation (P < 0.0001; Student’s t test).