Figure 1. Interaction of Anks1a with EphA2 is involved in the transport of EphA2 from the ER to the plasma membrane.

(a) CT26 cells were stained with an anti-Anks1a antibody. Subcellular organelles were detected with ER markers (Calnexin and Calregulin) or Golgi markers (GM130 and p230). Scale bar, 10 μm. (b) Anks1a co-localization with the ER or Golgi was calculated using Imaris (n=15 for each antibody). ***P<0.001, one-way analysis of variance (ANOVA) test. (c) MEF cells from wild-type or Anks1a-null mutant mice were incubated with 1 μg ml⁻¹ ephrinA5-Fc at 4 °C for 1 h and ephrinA5-Fc at the surface was visualized via fluorescein isothiocyanate (FITC)-conjugated anti-human IgG. Scale bar, 25 μm. (d) The percentage of ephrinA5-Fc-bound cells (n=25 for both WT and knockout (KO) MEFs). (e) Total fluorescence of cell-surface-bound ephrinA5-Fc was calculated using ImageJ (n=25 for both WT and KO MEFs). Data represent means±s.e. ***P<0.001, Student's t-test. (f) VC-tagged EphA2 was co-transfected into HEK293T cells with VN-tagged Anks1a constructs. Cells were fixed and analysed for BiFC signals. Scale bar, 20 μm. (g) HEK293T cells were transfected with both VN-tagged Anks1a and VC-tagged EphA2 before being stained with the indicated subcellular markers. Scale bar, 10 μm. (h) Co-localization efficiency for the Anks1a/EphA2 complex with each marker was calculated using Imaris (n=20 for each transfection). *P<0.05, one-way ANOVA test. (i) Anks1a KO MEF cells were transfected with the indicated Anks1a constructs. Surface-binding assays were performed 48 h after transfection as described in c. The transfected cells were detected by an anti-GFP antibody that recognizes the VN portion fused to each Anks1a protein. Scale bar, 20 μm. (j) Fluorescent intensity was calculated as described in b (n=15 for each transfection). **P<0.01, one-way ANOVA test. Data represent means±s.e.