SS-GFP-Lys is transported together with podocalyxin to the apical membrane. (A) EpH4 cells stably expressing SS-GFP-Lys were transfected with one of GPI-mScarlet, E-cadherin-mCherry, or Podocalyxin-like-1-mScarlet (PODXL1-mScarlet). Living cells were observed with a confocal microscope at 37°C. GPI-mScarlet and PODXL1-mScarlet showed co-localization with SS-GFP-Lys dots, but E-cadherin-mCherry did not. Scale bar, 5 μm. (B) The degree of co-localization between SS-GFP-Lys and one of GPI-mScarlet, E-cadherin-mCherry, or PODXL1-mScarlet was analyzed by Pearson’s correlation coefficient in colocalization (ImageJ). N = 3 independent experiments; error bar, SD; ***, P = 0.0005; *, P(Podocalyxin-like-1) = 0.0143; and *, P(E-cadherin) = 0.0170 by one-way ANOVA with Tukey’s post-hoc test. (C) EpH4 cells stably expressing SS-GFP-Lys were cultured on a two-chamber filter and grown to confluence. After 48 h of culture, the medium of the apical side and that of the basolateral side were collected and concentrated 30-fold. (D) Samples obtained as in Fig. 3 C were resolved by SDS-PAGE and immunoblotted with anti-GFP mAb. Arrowhead indicates the expected molecular weight of SS-GFP-Lys. Source data are available for this figure: SourceData F3.