Human cervical cancer HeLa cells and human embryonic kidney (HEK) 293 T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For the luciferase assay, HEK293T cells (105) were transfected with 1 μg pCAGIG-V5 vectors encoding Vax1, Vax1AA, or Vax1(R152S) cDNA together with pGL3-Tcf7l2-luciferase (0.2 μg) and pCMV-β-gal (0.2 μg) reporter constructs. Luciferase activity in the transfected cells was measured at 24 h posttransfection and normalized to β-galactosidase activity to obtain the relative luciferase activity of the cells. To test the cellular penetration of the Vax1 protein, V5 peptides or V5-Vax1 proteins, which were purified from HEK293T cells, were added to the growth media (1.5 pmol/ml [final concentration]) of HeLa cells, which express GFP-Sdc2. The distribution of V5 peptides and V5-Vax1 proteins on the cell surface and inside the cells was examined by immunostaining with mouse anti-V5 and chicken anti-GFP antibodies.
Retinal explants were prepared as described previously16 . Briefly, the retina was prepared from mouse embryos at E13 and mixed with collagen in DMEM supplemented with 10% FBS. The retinal explants in collagen were then cultured in neurobasal medium containing B27 supplement (Invitrogen Inc.) for 48 h to allow the axons to grow from the explants. 6X-Histidine peptides or Vax1-6X-His proteins, which were purified from E. coli, were then added to the culture medium (2 pmol/ml [final concentration]) of retinal explants. Alternatively, the retinal explants were placed next to collagen droplets containing HEK293 cells (105 cells/droplet), which express Vax1 or Vax1AA. The lengths of retinal axons grown from the explants were measured before and after the treatments to determine the axon growth rate.
Retinal explants were prepared as described previously16 . Briefly, the retina was prepared from mouse embryos at E13 and mixed with collagen in DMEM supplemented with 10% FBS. The retinal explants in collagen were then cultured in neurobasal medium containing B27 supplement (Invitrogen Inc.) for 48 h to allow the axons to grow from the explants. 6X-Histidine peptides or Vax1-6X-His proteins, which were purified from E. coli, were then added to the culture medium (2 pmol/ml [final concentration]) of retinal explants. Alternatively, the retinal explants were placed next to collagen droplets containing HEK293 cells (105 cells/droplet), which express Vax1 or Vax1AA. The lengths of retinal axons grown from the explants were measured before and after the treatments to determine the axon growth rate.
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