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. 2017 Mar 6;216(3):743–760. doi: 10.1083/jcb.201607095

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© 2017 Badgandi et al.

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Figure 7. — TULP3/TUB-mediated trafficking to ciliary membrane. (A) IMCD3 cells stably expressing ^SNAPGpr161^GFP were sequentially transfected with 200 nM of the indicated siRNAs twice as indicated in the experimental format on the top left (see Materials and methods). Starved cells were blocked with SNAP–surface block for 30 min, washed, and then were treated with SNAP–surface 594 at indicated time points. Cells were fixed and immunostained for GFP (green) and acetylated tubulin (magenta). Cilia positive and negative for SNAP–surface 594 are marked by white arrows and arrowheads, respectively. The yellow arrowhead points to cilia faintly stained for SNAP–surface 594. Total counted cells are >150 for control and >200 for Tulp3 siRNA, respectively. Data represent means ± SD from two or more fields from a single experiment. Bars, 5 µm. (B) NIH 3T3 Flp-In cells stably expressing Gpr161-GFP or Gpr161^V158E-GFP mutants were sequentially transfected with 200 nM of indicated siRNAs twice for 72 h (see Materials and methods) and serum starved for the last 24 h before fixing and immunostaining for GFP, acetylated tubulin, and DNA. Ciliary pixel intensities for GFP are shown. Total counted cells were >60 per condition from two independent transfections with >30 cells counted per coverslip. A.U., arbitrary units. Also see Fig. S5 E. (C) IMCD3 Flp-In cells stably and inducibly expressing ^MycTULP3 N terminus (1–183 aa) were starved in the presence of doxycycline (Dox) for 20 h (4 µg/ml). After washing, cells were treated ± SAG (500 nM) for indicated time points in starvation medium before fixing and immunostaining for Gpr161, Myc, acetylated tubulin, and DNA. Myc-positive and -negative cells were scored for Gpr161-positive cilia. Total counted cells are >55 and >180 for Myc-positive and -negative cells, respectively, for each time point. Data represent means ± SD from three coverslips. Also see Fig. S5 F. (A–C) *, P < 0.001 with respect to corresponding siRNA control at each time point measured (A); *, P < 0.0001 with respect to corresponding controls (B); *, P < 0.05; **, P < 0.01 with respect to corresponding 0 h time point (C). (D) Model for TULP3/TUB-mediated trafficking to ciliary membrane. See Discussion. BB, basal body; FP, fusion protein; NT, TULP3/TUB IFT-A binding N-terminus domain. Also see Fig. S5.