Fig. 1: Identification of a GAG sugar-binding motif of Vax1.

a Consensus amino acid sequences of GAG sugar-binding motifs in mouse Otx2 and Vax1. b Expression of V5-Vax1 and EGFP, which are independently translated from the same transcript, was examined by immunostaining transfected HeLa cells with mouse anti-V5 (red) and chicken anti-GFP (green) antibodies. Arrows indicate HeLa cells expressing V5-Vax1 without EGFP, implicating the transfer of V5-Vax1 but not EGFP from V5;EGFP double-positive cells. c HEK293T cells were transfected with a DNA vector encoding Vax1, Vax1(R152S), or Vax1(KR/AA) cDNA together with a Vax1 target Tcf7l2-luciferase reporter DNA construct. Luciferase activities in the transfected cells were measured at 24 h posttransfection. The values are averages obtained from four independent experiments, and error bars denote standard deviations (SDs). P values were determined by ANOVA (*, p < 0.01; **, p < 0.005; ***, p < 0.001; ns, not significant). d V5-tagged Vax1 or Vax1^AA proteins were expressed in HEK293T cells, and growth media of the transfected cells were then collected after incubating for 3 h in the presence (+) or absence (-) of heparin (10 mg/ml final; see Methods for details). Cell lysates and TCA-precipitated fractions of growth medium were analyzed by 10% SDS‒PAGE and subsequent western blotting (WB) with anti-Vax1 antibody (α-Vax1). The graph below the WB data shows the relative intensities of the Vax1 bands in the blots. The values are averages obtained from four independent experiments, and error bars denote the SD. e Interactions of V5-Vax1 and V5-Vax1^AA with GFP-Sdc2 in HEK293T cells were assessed by immunoprecipitation (IP) with α-V5 and subsequent WB with α-GFP. Relative amounts of V5-Vax1 and GFP-Sdc2 in the cell lysates were also examined by WB. (f) V5-Vax1 or V5-Vax1^AA recombinant proteins were added to the growth medium of HeLa cells expressing GFP-Sdc2 and incubated for 3 h. Vax1 proteins inside cells and/or at the cell surface were detected by immunostaining with mouse α-V5 (red) and chick α-GFP (green). g Retinas were isolated from E13.5 mice and cultured as described in the Methods. The axonal lengths of retinal explants were measured at 24 h postculture; then, the explants were treated with 6X-His-tagged recombinant Vax1 or Vax1^AA proteins. (i) Alternatively, retinal explants were cocultured with HEK293T cells transfected with pCAGIG (mock), pCAGIG-V5-Vax1, or pCAGIG-V5-Vax1^AA. Axonal lengths were remeasured after 24 h, and the explants were immunostained with α-Vax1 (green) and α-NF160 (red). Arrowheads indicate the areas magnified in each inset. h and j The changes in axonal length during the 24-h incubation period are shown in graphs. The values in the graph are averages, and error bars denote SDs (n = 6).