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. 2019 Jun 18;17:101–118. doi: 10.1016/j.isci.2019.06.020

Figure 5.

Figure 5

Dimerization and Vesiculation Ability of ANKHD1

(A) Illustration of human ANKHD1 and the fragments: ANKHD1 (1–2542 aa), N + ARD15 (ANKHD1 1–885 aa), ARD10 (ANKHD1 886–1587 aa), N + ARD25 (ANKHD1 1–1587 aa), and ARD25 (ANKHD1 195–1418 aa). ANK, amphipathic helix, and the KH domain were indicated. Disorder probability was also plotted by PrDOS (http://prdos.hgc.jp).

(B) Level of expressed ANKHD1 fragments, endogenous EEA1, and endogenous ANKHD1 in ANKHD1-depleted HeLa cells expressing EGFP, N + ARD15-EGFP, ARD10-EGFP, N + ARD25-EGFP, ARD25-EGFP, or ANKHD1-EGFP for rescue experiments by western blot. See also Figure S9.

(C) Quantification of EEA1 in (B). Data represent the mean of three independent experiments.

(D) Vesiculation by liposome sedimentation assay for N + ARD15, ARD10, N + ARD25, and ARD25 at 250 nM using ultracentrifugation at 109,000 × g. The lipid composition of the liposomes was PC:PE:PS:rhodamine-PE at weight ratio of 6:3:1:0.02.

(E) Quantification of liposomes in (D). Data represent the mean of three independent experiments.

(F) Electron micrographs of liposomes of PC:PE:PS at a weight ratio of 6:3:1 after incubation with N + ARD15, ARD10, N + ARD25, or ARD25 at 250 nM for 20 min. Liposomes were visualized by negative staining. The boxed region presents an enlarged image. Scale bars, 100 nm.

(G) Vesiculation by liposome sedimentation assay for N + ARD15 and ARD10 at 1 μM using ultracentrifugation at 109,000 × g. The lipid composition of the liposomes was PC:PE:PS:rhodamine-PE at weight ratio of 6:3:1:0.02.

(H) Quantification of liposomes in (G). Data represent the mean of three independent experiments.

(I) Cross-linking assay for ANKHD1 fragments. N + ARD15, ARD10, N + ARD25, or ARD25 at 250 nM were incubated with or without liposome composed of PC:PE:PS at a weight ratio of 6:3:1 and then incubated with BS(PEG)5 at a final concentration of 100 μM. The box indicates the dimer formation of ANKHD1 fragments.

All error bars represent SE. *p < 0.05, **p < 0.01, and ***p < 0.001. Statistical significance was determined with the Student's t test. ns, not significant.