Roles of IFT-A and BBSome in ciliary entry and exit. (A) Tulp3 is required for ciliary entry of SSTR3. Box plots of ciliary ^APSSTR3^NG intensities measured by NG fluorescence (RFUs) for various IMCD3 lines. Tulp3^−/− cells were fixed to identify cilia using antiacetylated tubulin staining, and all other cells were imaged live as cilia were readily identified in the NG channel. NG fluorescence was not affected by fixation (Fig. S2 B). Asterisks indicate ANOVA significance values. ***, P < 0.0001; n.s., P > 0.05. n = 18–59 cilia. (B) IFT-A directly recognizes SSTR3ⁱ³. The IFT-A complex was purified from IMCD3-[LAP-IFT43] cells and incubated with beads coated with GST-SSTR3ⁱ³ or GST-SSTR5ⁱ³. Captured materials were eluted by cleaving off the beads and visualized by silver stain and immunoblotting. Five input equivalents were loaded in the eluate lanes. (C) Model of ciliary entry. (D and E) BBSome subunits were depleted by siRNA; Arl6, Ift27, and β-arrestin 2 (Arrb2) genes were knocked out by genome editing; and SA/TA denotes a phosphomutant of the C tail of SSTR3 that is unable to bind to β-arrestin 2 (Roth et al., 1997). (D) Representative time series of ^APSSTR3 pulse-labeled with mSA647 under different conditions. Bar, 2 µm. (E) Absolute retrieval rates were calculated by linear fitting of retrieval kinetics measured form SSTR3 pulse-chase labeling as in D (see Materials and methods; Nager et al., 2017). Error bars represent error of the fit. n = 10–35 cilia. (F) BBSome purified to near-homogeneity from bovine retina was incubated with glutathione beads coated with GST, GST-GPR161^Ct, and GST-SSTR3ⁱ³. Captured materials were cleavage eluted and immunoblotted. Three input equivalents were loaded in the eluate lanes. MW, molecular weight. (G) Signal-dependent retrieval requires the joint activities of Arl6-GTP, BBSome, and β-arrestin 2.