Supplemental Materials

This article contains the following supporting material:

• Supplementary Figure-1 - FRT cells redistribute LDLR-GFP from intracellular compartments to the basolateral cell surface during incubation in serum-free medium. FRT cells permanently transfected with LDLR-GFP plasmid vector were grown on coverlips until 2 days post-confluency and then were incubated either in the presence or absence of 5% FBS for 3 h at 37ºC. In the presence of serum, the LDLR-GFP distributes mainly in intracellular compartments, although being also detectable at the basolateral cell surface. In contrast, in the absence of serum it redistributes in most of the cells to the basolateral cell surface. Scale bar 10 μm.
• Supplementary Figure-2 - Polarity of recently confluent FRT cells.
  FRT cells were grown on coverslips until reaching confluency and after 2 days post-confluency they were intranuclearly microinjected with plasmid vectors encoding LDLR_Y18A-GFP (endocytosis-defective mutant), VSVG-GFP, TfR-GFP or the apical VSVG version VSVG3-GFP. The cells were then incubated for 1 h at 37ºC in DMEM-HEPES supplemented with 5% FBS to allow sufficient expression as to detect the fluorescencent cargos. Then, the medium was replaced by serum-free medium supplemented with CHX (100 μg/ml) for the rest of the experiment. After a TGN block for 2 h at 20ºC, transport was reestablished by shifting the temperature to either 32ºC for VSVG or 37ºC for the other cargos. After1.5 h the cells were fixed and treated for indirect immunofluorescence for the tight junction marker ZO-1. Confocal fluorescent images were processed with Metamorph software. Basal, lateral and apical focal planes are shown. LDLR, VSVG and TfR are seen mainly at or below the plane of ZO-1. The VSVG3 is only apically detected, but its low level of expression does not allow to discarding a minor proportion in the basolateral domain. Scale bar 10 μm.