"We precipitated endogenous proteins using immobilized mouse and human SF-TAP-tagged PIFO in ciliated and cilia-disassembling HEK293T cells. This pull-down analysis revealed that the early endosome marker Rab6, the late endosome marker Rab8, the cilium localized small GTPase Arl13b (Caspary et al., 2007), the PCM protein γ-Tubulin, the centriolar protein Centrin, the MT protein β-Tubulin, and the motor protein Kif3a, all physically interact with Pifo/PIFO in cell culture (Figure 5G and Figures S5E–S5H)."

"When we acutely ablated Talpid3 using several distinct siRNAs in RPE1 cells and induced cilia formation by serum starvation, we found a significant reduction in primary cilia assembly using four canonical markers, glutamylated tubulin (GT335), detyrosinated tubulin, IFT88, and Arl13b (Fig. S2, B and C; and unpublished data)."

"Furthermore, we immunoprecipitated Arl13b from nonciliated HeLa cells and found that Sec8 and Sec5 are also enriched in the precipitates in a GTPγS-sensitive manner, indicating that the interaction is conserved and not dependent upon the presence of cilia (Supplemental Figure S1)."

"Finally, we were able to purify human Arl13B (18–278) from insect cells in reasonable amounts to test its GEF activity. Confirming the conservation of structure and function the human Arl13B also exhibits strong GEF activity for Arl3 (Figure 6A)."

"We found the GAP-dependent GTP hydrolysis was Arl13b-dependent, indicating that it is a GAP and not a nonspecific nucleotidase."

"However, given that all preparations yielded very similar rates of intrinsic GTP hydrolysis and every purified protein preparation contains small amounts of contaminants, we cannot currently exclude the possibility of contaminating, nonspecific nucleotidase(s) being responsible for these weak activities, despite the use of the GST-only negative control."

"To distinguish between a direct or indirect association of ARL13B with tubulin, we isolated recombinant GST-tagged human ARL13B from E. coli and incubated it in the presence of purified bovine brain tubulin. Incubation of the GST protein tag with tubulins was used as control. Glutathione (GSH) sepharose beads were added to each reaction mix to purify GST-ARL13B or control GST, and their interacting proteins were analyzed by western blotting. Tubulin was found in the eluate from the beads loaded with GST-ARL13B, but not in that from control GST ( Fig. 2 A and B, lanes ‘GST-ARL13B aa 1-428’ and ‘GST’), thus indicating that ARL13B can directly interact with tubulin dimers."

"However, the ectopic expression of GFP-tagged wild-type mARL13B or huARL13B re-established a cilia length of most Arl13b hnn MEFs to be 2.0–4.0 µm ( Fig. 3 E,F) ( Larkins et al., 2011 )."

"In contrast, in Arl13b hnn MEFs SMO concentrated in discrete regions of the cilium located mostly at distal and/or proximal extremities of cilia regardless the presence of SHH in the medium ( Fig. 5 B). However, we noticed that SMO in cilia of SHH-stimulated Arl13b hnn MEFs was almost exclusively detected at the distal tip of the cilium ( Fig. 5 B,E). Similarly, the mCherry-SSTR3 also failed to distribute evenly along the cilium and accumulated at the distal tip of Arl13b hnn MEFs cilia ( Fig. 5 H)."

"We therefore wanted to test the effect of BART on ARL3 activation; the addition of BART to the ARL13B-mediated reaction is twofold: a significant amount of mantGDP is displaced, and the ARL3 nucleotide exchange rate is markedly increased (Figure 3B). Titrating in BART to the ARL13B-mediated reaction resulted in a maximum increase of over 500-fold that of the intrinsic rate of GDP dissociation (Figure 3C)."