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. 2022 Sep 21;221(10):e202111076. doi: 10.1083/jcb.202111076

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© 2022 Liu et al.

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Figure 2. — ARL3^GDP binds the membrane via its N-terminal amphipathic helix for diffusing into cilia. (A) Schematic presentation of bacterially expressed ARL3 and its variants (shown on the left). ΔN15 stands for the N-terminal 15 amino acids of ARL3 deleted. Immunoblots of liposome flotation captured ARL3 preloaded with GTPγS or GDP (shown in the middle) and its variants (shown on the right) with α-ARL3. ARL3 or its variants along was used as an input for evaluating their binding/input ratio shown as percentile. (B) Immunoblots of WC samples and cilia of cells indicated on the top probed with α-ARL3. α-Tubulin and acetylated (Ac)-tubulin were used to adjust the loading of WC samples and cilia, respectively. MW, molecular weight. (C) Representative TIRF images and corresponding kymograms of cells indicated on the top (Videos 2, 3, and 4, 15 fps). The time and transport lengths are indicated on the right and on the bottom, respectively. The ciliary base (base) and tip (tip) were shown. (D) Immunoblots of ciliary fractions of cells indicated on the bottom probed with α-HA, α-IFT57 (ciliary matrix marker), α-PLD (ciliary membrane marker) and Ac-tubulin (axoneme marker). (E) Schematic presentation of how ARL3^GDP binds the membrane for diffusing into cilia prior to converting to ARL3^GTP for being enriched at the proximal ciliary region. Source data are available for this figure: SourceData F2.