Figure 2.

Localization and expression of Cx43, adherens and tight junction proteins, and cytoskeletal proteins in RL-EΔN and RL-NΔE cells. (A) Western blot analysis of the expression level of E-Cad (E-cad) and N-Cad (N-cad), Cx43, α- and β-catenins, occludin and ZO-1, cytokeratin 8 and vimentin and actin. Note that RL-EΔN cells express only E-Cad, but no detectable level of N-Cad, whereas RL-NΔE cells express only N-Cad but no E-Cad. Note also that the expression level of Cx43 and other proteins is not discernibly different in the two cell lines. (B) RL-EΔN and RL-NΔE cells were seeded on glass coverslips and immunostained for Cx43, E-Cad (E-cad), N-Cad (N-cad), and occludin (Occl). Note that Cx43 is assembled into GJs in RL-EΔN cells, whereas it remains intracellular as vesicular puncta in RL-NΔE cells. Note also that E-Cad, N-Cad, and occludin are localized at the areas of cell–cell contact in both cell types. (C) RL-EΔN and RL-NΔE cells were seeded on transwell filters for 5 d and immunostained for Cx43 and ZO-1. Note a more robust expression of ZO-1 at cell–cell contact areas in RL-EΔN cells compared with RL-NΔE cells. Note also partial overlap of cell boundaries in RL-NΔE cells as well as GJ like punctate structures. Nuclei (Blue) were stained with DAPI. Bar = 10 μm in B and 15 μm in C.