Figure 3A. Diagrams of mouse Rabl2 and its GTP (Q80L)- or GDP (S35N)-locked mutants. B. Subcellular localization of Rabl2 and mutants. RPE1 cells infected with adenovirus to express the GFP-tagged proteins were serum-starved for 48 h to induce ciliogenesis. Ac-tub served as ciliary marker. Arrowheads indicate ciliary base. The cells were used for quantifications in (C) and (D). C. Reduced ciliary base localization of GFP-Rabl2S35N as compared to GFP-Rabl2. Results from three independent experiments (n=30 for each experiment and condition) were pooled together. D. GFP-tagged Rabl2 and its mutants did not significantly affect ciliogenesis upon overexpression as compared to uninfected control cells. At least 104 cells were scored in each experiment and condition. E. Rabl2Q80L retained ciliary GPR161 after SAG stimulation. RPE1 cells infected as in (B) were serum-starved for 24 h, followed by DMSO (mock) or SAG treatment for 24 h. Arrows indicate cilia that are displayed in insets. At least 100 cilia were scored in each experiment and condition. F. Rabl2Q80L led to SAG-independent accumulation of ciliary Smoothened (SMO). RPE1 cells were treated as in (E). ARL13B served as ciliary marker. The white arrows point to positions of cilia. At least 100 cilia were scored in each experiment and condition. Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunostained to examine ciliary Gli2 The white arrowheads (G) point to ciliary tips. Quantification results were from three independent experiments, each using MEFs from different Rabl2-deficient embryos. Gli2 intensities, presented in arbitrary unit, were measured from at least 22 cilia in each experiment and condition and pooled together. H. Rabl2Q80L inhibited Hh signaling. MEFs from Rabl2-/- E12.5 embryos infected with lentivirus to express GFP, GFP-Rabl2 or GFP-Rabl2Q80L were serum-starved for 24 h and treated with DMSO (mock) or 100 nM SAG for an additional 24 h. Uninfected MEFs from the wildtype littermate embryos served as the positive control. The samples were immunoblotted to examine levels of Gli1 and Gli3 In the samples for immunoblotting (H), GFP-positive cells occupied 82.3% (GFP), 87.3% (GFP-Rabl2), and 81.2% (GFP-Rabl2Q80L), respectively, when 260 cells in each sample were scored. Relative band intensities are shown in red. I. A model summarizing the results in Fig 3 (left half) and speculating a switch role of endogenous Rabl2 for ciliary GPCR export and signaling, taking the Hh signaling as an example (right half). The GTP-locked Q80L mutant represses the export of ciliary Gpr161 and Smo, leading to reduced Gli-A and possibly increased Gli-R formations and accordingly attenuated Gli1 expression. Endogenous ciliary Rabl2-GTP would similarly repress the export of Gpr161 and Smo until it was converted to GDP-binding form through GTP hydrolysis. Proper regulation of the GTP hydrolysis would therefore facilitate proper Hh signaling.