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. 2017 Jul 3;216(7):2131–2150. doi: 10.1083/jcb.201611138

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© 2017 Monis et al.

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Figure 1. — IFT20 knockdown affects fibrocystin and polycystin-2 ciliary trafficking but only modestly affects smoothened trafficking to the primary cilium. Quantification of (A) CD8-fibrocystinCTS-SNAP, (B) polycystin-2(1-703)-GFP-SNAP, and (C) smoothened-SNAP-GFP trafficking from the Golgi apparatus to the primary cilium during lenti-shRNA knockdown of IFT20. (A–C, subpanel a) Selected immunoblot images of IFT20 knockdown and γ tubulin loading control. (A–C, subpanel b) Quantification of knockdown. Mean protein levels plotted from three independent experiments for each condition (control [Con.], NS-shRNA [NS.], shRNA#1, and shRNA#2) and normalized to their corresponding γ tubulin loading control. shRNA-mediated knockdown of IFT20 results in >90% reduction of total protein abundance. (A–C, subpanel c) Mean cilia length, (A–C, subpanel d) mean CD8 or GFP ciliary fluorescence, and (A–C, subpanel e) mean SNAP ciliary fluorescence plotted from three independent experiments in which 30 cilia were quantified for each condition (control, NS-shRNA, shRNA#1, and shRNA#2) at each time point (n = 90 total cilia per time point). CD8 or GFP and SNAP pixel intensity measurements were taken at individual time points starting at the time of temperature shift from 19°C to 37°C (0 h). Data were analyzed using one-way ANOVA and the Bonferroni multiple-comparisons test. The control condition was compared with the shRNA#1 and shRNA#2 conditions to determine statistical significance. (A–C, subpanel f) Linear regression analysis of selected time points of newly synthesized membrane protein delivery to the cilium was completed. Slope values between control and experimental shRNA groups were compared with one another to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent SEM.